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Fronto-limbic circuits in the primate brain are responsible for executive function, learning and memory, and emotions, including fear. Consequently, changes in gene expression in cortical and subcortical brain regions housing these circuits are associated with many important psychiatric and neurological disorders. While high quality gene expression profiles can be identified in brains from model organisms, primate brains have unique features such as Brodmann Area 25, which is absent in rodents, yet profoundly important in primates, including humans. The potential insights to be gained from studying the human brain are complicated by the fact that the post-mortem interval (PMI) is variable, and most repositories keep solid tissue in the deep frozen state. Consequently, sampling the important medial and internal regions of these brains is difficult. Here we describe a novel method for obtaining discrete regions from the fronto-limbic circuits of a 4 year old and a 5 year old, male, intact, frozen non-human primate (NHP) brain, for which the PMI is exactly known. The method also preserves high quality RNA, from which we use transcriptional profiling and a new algorithm to identify region-exclusive RNA signatures for Area 25 (NFκB and dopamine receptor signaling), the anterior cingulate cortex (LXR/RXR signaling), the amygdala (semaphorin signaling), and the hippocampus (Ca++ and retinoic acid signaling). The RNA signatures not only reflect function of the different regions, but also include highly expressed RNAs for which function is either poorly understood, or which generate proteins presently lacking annotated functions. We suggest that this new approach will provide a useful strategy for identifying changes in fronto-limbic system biology underlying normal development, aging and disease in the human brain.

Reverse phase protein array (RPPA) technology introduced a miniaturized “antigen-down” or “dot-blot” immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

The most common mutation in cystic fibrosis (CF) is DeltaF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked DeltaF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of DeltaF508-CFTR. The half-life of DeltaF508-CFTR is also dramatically enhanced. Dexamethasone-activated DeltaF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and DeltaF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects DeltaF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated DeltaF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.

In a previous study of sodium 4-phenylbutyrate (4-PBA)-responsive proteins in cystic fibrosis (CF) IB3-1 bronchial epithelial cells, we identified 85 differentially expressed high abundance proteins from whole cellular lysate (Singh, O. V., Vij, N., Mogayzel, P. J., Jr., Jozwik, C., Pollard, H. B., and Zeitlin, P. L. (2006) Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells. J. Proteome Res. 5, 562–571). In the present work we hypothesize that a subset of heat shock proteins that interact with cystic fibrosis transmembrane conductance regulator (CFTR) in common during chemical rescue and genetic repair will identify therapeutic networks for targeted intervention. Immunocomplexes were generated from total cellular lysates, and three subcellular fractions (endoplasmic reticulum (ER), cytosol, and plasma membrane) with anti-CFTR polyclonal antibody from CF (IB3-1), chemically rescued CF (4-PBA-treated IB3-1), and genetically repaired CF (IB3-1/S9 daughter cells repaired by gene transfer with adeno-associated virus-(wild type) CFTR). CFTR-interacting proteins were analyzed on two-dimensional gels and identified by mass spectrometry. A set of 16 proteins known to act in ER-associated degradation were regulated in common and functionally connected to the protein processing, protein folding, and inflammatory response. Some of these proteins were modulated exclusively in ER, cytosol, or plasma membrane. A subset of 4-PBA-modulated ER-associated degradation chaperones (GRP94, HSP84, GRP78, GRP75, and GRP58) was observed to associate with the immature B form of CFTR in ER. HSP70 and HSC70 interacted with the C band (mature form) of CFTR at the cell surface. We conclude that chemically rescued CFTR associates with a specific set of HSP70 family proteins that mark therapeutic interactions and can be useful to correct both ion transport and inflammatory phenotypes in CF subjects.

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47–374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously.

Cystic fibrosis (CF) is a common, lethal genetic disease, which is due to mutations in the CFTR gene. The CF lung expresses a profoundly proinflammatory phenotype, due to constitutive hypersecretion of IL-8 from epithelial cells lining the airways. In a systematic search for candidate drugs that might be used therapeutically to suppress IL-8 secretion from these cells, we have identified a potent and efficacious series of amphiphilic pyridinium salts. The most potent of these salts is MRS2481, an (R)-1-phenylpropionic acid ester, with an IC50 of ca. 1microM. We have synthesized 21 analogues of MRS2481, which have proven sufficient to develop a preliminary structure-activity relationship (SAR). For optimal activity, we have found that the ester must be connected to the pyridinium derivative by an eight-carbon chain. An optical isomer of the lead compound, containing an (S)-1-phenylpropionic acid ester, has been found to be a much less active. The mechanism of action of MRS2481 appears to involve inhibition of signaling of the NF(kappa)B and AP-1 transcription factors to the IL-8 promoter. MRS2481 is a potent inhibitor of TNFalpha-induced phosphorylation and proteosomal destruction of I(kappa)B(alpha). Inasmuch as I(kappa)B(alpha) is the principal inhibitor of the NF(kappa)B signaling pathway, preservation of intact I(kappa)B(alpha) would serve to keep the IL-8 promoter silent. We also find that MRS2481 blocks TNF(alpha)-activated phosphorylation of JNK, the c-JUN kinase. The IL-8 promoter is also activated by an AP-1 site, which requires a phospho-c-JUN/c-FOS dimer for activity. We therefore interpret these data to suggest that the mechanism of MRS2481 action is to inhibit both NF(kappa)B and AP-1 signaling on the IL-8 promoter. Given the medicinally promising properties of water-solubility, potency in the low muM concentration range, and high efficacy, we anticipate that MRS2481, or a further optimized derivative, may find an important place in the armamentarium of pharmaceutical strategies yet to be arrayed against the inflammatory phenotype of the CF lung.

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.

Loss of heterozygosity (LOH) in the 10q21 region that harbors the tumor suppressor gene ANX7 -GTPase gene have been found in 35% of prostate tumors. Therefore, the rationale for this study is that this gene could also be implicated in breast pathogenesis as well. We investigated allelic losses in microsatellites of the 10q21 region, and their correlations with ANX7 status, estrogen receptor (ER) status, progesterone receptor (PR) status, Ki-67 status and pathological phenotype in 30 breast carcinomas with matched control specimens. The LOH analysis was performed by amplifying DNA by PCR, using four markers of the 10q21 region (AFMa299ya5, AFM220xe5, AFM 063xc5, AFM200wf4). LOH in at least one marker of the 10q21 region (AFM220xe5 marker close to ANX7) was found in 66% of the first set of informative tumors containing 10 pairs of specimens. Subsequent comparison between 20 carcinomas using AFM220xe5, with and without LOH in terms of pathological parameters showed significant associations with differences in age ( P =0.04), ER ( P =0.05), Ki-67 ( P =0.04) and PR ( P =0.01); a trend toward significance was found for tumor size ( P =0.06) and histological grade III ( P =0.14). These results suggest that the ANX7 gene, or other genes of the 10q21 region, could be functionally related to breast cancer, probably influencing the hormone receptor expression associated with poor prognosis during development.

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.

Annexins are a gene family of Ca2+-dependent membrane binding proteins which interact specifically with acidic phospholipids. We describe here details of highly sensitive and precise assays for annexins I and V, utilizing turbidometric analysis of phosphatidylserine liposome aggregation. In the case of annexin I, the new assay is 7-fold more sensitive than the conventional chromaffin granule aggregation assay in terms of threshold for detection and the rate of increase of initial absorbance is 15-fold greater. Annexin V, which binds but does not aggregate liposomes, can be assayed on the basis of inhibition of Ca2+-dependent liposome aggregation. Further comparative advantages of the assay include lower expense and increased shelf life of the liposome reagent.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce a parkinsonian syndrome in humans and nonhuman primates, which is susceptible to treatment and prevention by drugs such as L-DOPA and L-deprenyl. Recently, we have reported that MPTP can also cause a parkinsonian syndrome in the common goldfish, which appears to faithfully mirror the neurochemical and behavioral aspects of the action of MPTP in the higher vertebrates. In addition, we recently identified the likely teleost equivalent of the substantia nigra in the goldfish forebrain, the "nucleus pars medialis," on the basis of its destruction by MPTP and selective protection by the MAO-B blocker L-deprenyl. In the present work we substantiate this conclusion by examining tissue destruction the goldfish forebrain at increasing MPTP concentrations, up to the the LD50 of 200 mg/kg. In addition, we show that at the highest MPTP dose subtle changes also occur with low frequency in nondopaminergic cells in the optic tectum, and in ependymal cells lining the midbrain ventricle. The effects on ependymal cells are similar to those previously noted in the forebrain. We conclude that the goldfish model continues to faithfully mimic the histologic pattern of parkinsonian tissue destruction engendered by MPTP in primate models.

Several lines of evidence suggest that annexins, a family of phospholipid-binding proteins, play a role in cellular trafficking. Five annexins (I, II, V, VI, VII) were detected in rat adipose cells. They were primarily associated with the plasma membrane in a calcium-dependent manner. None of them redistributed with insulin treatment of the cells, in contrast to the glucose transporter GLUT4, which moved from intracellular membranes to the plasma membrane. Although the actual function of annexins in adipose cells remains to be determined, our data indicate that insulin-stimulated GLUT4 trafficking does not rely on a change in subcellular location of any of the five annexins detected so far in these cells.

The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinsonian syndrome in the goldfish (Carassius auratus), characterized by transient bradykinesia, the accumulation of MPP+ in the brain, and a decrease in the forebrain and midbrain content of catecholamines (Pollard et al., FASEB J., 6 (1992) 3108-3116). Using light and electron microscopy, we studied the effect of MPTP on the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive, dopaminergic neurons, and on the ultrastructure of other selected areas of the goldfish brain. Goldfish were treated with MPTP (50 mg/kg) in the absence or presence of L-deprenyl (10 mg/kg) or clorgyline (10 mg/kg). In the medial part of the central telencephalon, the nucleus telencephali, pars medialis, MPTP caused a decrease in the number of TH-immunoreactive neurons and distortions in their labelling pattern. Electron microscopic observations showed that MPTP caused swelling of cell processes, changes in neuronal nuclear profiles, dilation of endoplasmic reticulum, intracellular vacuolization and membrane distortions, and degeneration of neuronal fibers in this brain area. MPTP also caused a small reduction and some diffuseness in the labelling of dopaminergic neurons in several diencephalic periventricular nuclei. Moreover, MPTP induced cell swelling and degeneration in the subependymal cell layers along the forebrain ventricles. In all areas, L-deprenyl appeared to partially prevent the MPTP-induced degenerative changes. We conclude that in the goldfish MPTP causes marked histochemical changes in selected dopaminergic brain systems coincident with the Parkinson-like locomotor and neurochemical deficits.

Ion channels can express multiple conductance levels that are not integer multiples of some unitary conductance, and that interconvert among one another. We report here that for 26 different types of multiple conductance channels, all allowed conductance levels can be calculated accurately using the geometric sequence gn = g(o) (3/2)n, where gn is a conductance level and n is an integer > or = 0. We refer to this relationship as the "3/2 Rule," because the value of any term in the sequence of conductances (gn) can be calculated as 3/2 times the value of the preceding term (gn-1). The experimentally determined average value for "3/2" is 1.491 +/- 0.095 (sample size = 37, average +/- SD). We also verify the choice of a 3/2 ratio on the basis of error analysis over the range of ratio values between 1.1 and 2.0. In an independent analysis using Marquardt's algorithm, we further verified the 3/2 ratio and the assignment of specific conductances to specific terms in the geometric sequence. Thus, irrespective of the open time probability, the allowed conductance levels of these channels can be described accurately to within approximately 6%. We anticipate that the "3/2 Rule" will simplify description of multiple conductance channels in a wide variety of biological systems and provide an organizing principle for channel heterogeneity and differential effects of channel blockers.

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The possible physiological role of cytosolic pH changes in adrenal medullary chromaffin cell secretion was examined by investigating the effects of catecholamine secretagogues on cytosolic pH, which was monitored using the intracellular fluorescent indicator 2′,7′-bis-(2-car☐yethyl)-5(6)-car☐yfluorescein (BCECF). Anti-fluorescein antibodies were used to reduce background fluorescence from extracellular 2′,7′-bis-(2-car☐yethyl)-5(6)-car☐yfluorescein (BCECF). Stimulation with both cholinergic agonists (acetylcholine, nicotine) and a depolarizing agent (high K + ) transiently acidified the cytosol of the chromaffin cell. This acidification was antagonized by reducing extracellular Ca 2+ concentration and by Ca 2+ antagonists (Co 2+ , verapamil), indicating that it occurred secondarily to Ca 2+ influx, possibly as a result of exchange of Ca 2+ ions for protons across organelle membranes. Taken together with previously published data [Kuijpers G.A.J. et al . (1989) J. biol. Chem. 264 , 698–705] showing no effect of cytosolic acidification on nicotine-induced catecholamine secretion, these results indicate that secretagogue-induced cytosolic pH changes do not represent a causal step in stimulus-secretion coupling of the chromaffin cell. The cytosolic pH recovery to pre-stimulatory cytosolic pH levels was delayed by amiloride and by5-(N,N-dimethyl)amiloride, at concentrations that otherwise substantially inhibited cytosolic pH recovery from the rebound acidification induced by a 40-fold sudden dilution of NH 4 Cl. This latter type of recovery results from activation of an Na + /H + exchange mechanism. Therefore, the data suggest that Na + /H + exchange is actively involved in the dissipation of the small acid loads generated by secretagogues.

Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

Venom from the poison glands of the polychaete annelid Glycera convoluta has been reported to dramatically increase the frequency of miniature end-plate potentials at the frog and crayfish neuromuscular junctions, without causing detectable ultrastructural changes. We report here that addition of venom from the related annelid Glycera dibranchiata to one side of a lipid bilayer results in the formation of ion-permeable channels in the membrane. The channel forming activity was found in the void volume of a Sephadex G-25 column (mol. wt. greater than 5000). The conductance of a single channel is about 350 pmho in 0.1 M NaCl and is ohmic. The channels exhibit moderate (but not ideal) cation selectivity in NaCl or KCl gradients. Other selectivity measurements suggest that Ca2+ and Mg2+ are also permeable. The channels show a slight voltage sensitivity. The steady state conductance at--70 mV (side opposite venom) is about 5 times the conductance at + 70 mV. We suggest that these channels in the venom may evoke transmitter release at neuromuscular junctions either by (1) depolarizing the pre-synaptic terminal and thus opening voltage-dependent Ca2+ channels, or (2) directly allowing Ca2+ to enter the terminal. Black widow spider venom is known to produce similar effects on neuromuscular junctions and lipid bilayers. The single channel conductances and ionic selectivities of the channels found in the venoms of Glycera and Latrodectus are strikingly similar. Taken together, these results suggest that channel formation can explain the electrophysiologic effects of these two different venoms.

Three commonly used methods for the determination of epinephrine and norepinephrine levels in adrenal medullary tissue were compared. Two variations of the trihydroxyindole procedure, which utilized oxidation at room temperature or 0 degrees C, underestimated levels of total catecholamines in certain standard solutions and were unable to determine correctly their norepinephrine:epinephrine ratios. However, both the variability and the underestimation of the trihydroxyindole procedure carried out at room temperature were more pronounced than that of the trihydroxyindole assay at 0 degrees C. In addition, we tested an isocratic HPLC method utilizing electrochemical detection which separates epinephrine from norepinephrine. The ability of this method to measure correctly total and individual catecholamine levels was superior to either trihydroxyindole procedure, as was its variability. When the three assay methods were used to measure total and individual catecholamine levels in cultured adrenal bovine chromaffin cells, both the trihydroxyindole (0 degrees C) method and the HPLC method yielded values in agreement with those in the literature. However, the HPLC method produced data with lower error estimates. The trihydroxyindole (room temperature) assay was unable to reliably measure levels of epinephrine and norepinephrine in chromaffin cells. These comparisons of catecholamine assays demonstrated that there are circumstances under which the use of each is appropriate. In experiments where the epinephrine:norephinephrine ratio may be changing, the more accurate and precise HPLC assay may be essential, since the trihydroxyindole assays underestimate total catecholamines to varying degrees depending on this ratio. However, the HPLC method suffers from a requirement for technical sophistication for routine use. Therefore, in some laboratories and for repetitive measurement of many samples, the trihydroxyindole assay has a distinct advantage due to its easy utilization and ubiquitous materials. However, the superior results obtained with the trihydroxyindole (0 degrees C) assay over the trihydroxyindole (room temperature) assay emphasizes the need to evaluate the trihydroxyindole procedure for the required purpose, especially if differential oxidation is used for estimation of individual catecholamine levels.

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.

We have measured on-line the exocytotic secretion of ATP from adrenal medullary chromaffin cells induced by Ba 2+ using a luciferin/luciferase assay. We have found that Ba 2+ -induced ATP release requires the entry of Ba 2+ through either voltage- or receptor-gated Ca 2+ channels. This conclusion is based on the observations that short preincubations with low concentrations of either nicotine or K + greatly enhance Ba 2+ -induced ATP release and that this augmentation can be blocked with the nicotinic receptor antagonist, hexamethonium, and the Ca 2+ antagonist, nifedipine, respectively. Moreover, both nicotine and K 2+ stimulate 133 Ba 2+ uptake, which in the case of K 2+ is inhibited by nifedipine. These results support the hypothesis that the cellular events leading to Ba 2+ -induced secretion coincide at least in part with the events leading to Ca 2+ -dependent exocytosis.

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic 125 I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.

PC12 (phaeochromocytoma derived) cells possess the catecholamine synthesizing enzymes as well as the ability to store and release the catecholamines in response to K + . However, their monoamine oxidase activity and catecholamine release in response to tyramine has not been examined previously. PC12 cells have monoamine oxidase activity which oxidizes type A (noradrenaline and serolonin) and type A-B (dopamine, tyramine and kynuramine) substrates, and is selectively inhibited by clorgyline ( ic 50 ~ 10 −8 M ). In contrast, PC12 cell monoamine oxidase hardly oxidizes phenylethylamine, a type B substrate, and is relatively insensitive to inhibition by the selective monoamine oxidase type B inhibitor. I-deprenyl ( ic 50 ~ 10 −6 M ). By the above criteria it is apparent that the monoamine oxidase in PC12 is solely type A. The kinetics of the oxidase are similar to those of monoamine oxidase type A reported in other tissues including the adrenergic neuron, having apparent K m values of 400, 280, 170 and 227 μM for noradrenaline, dopamine, serotonin and tyramine. The apparent K m value for phenylethylamine is 235 μM. On the other hand, isolated chromaffin cells have the B form of monoamine oxidase with high affinity ( K m ~25μM) for phenylethylamine and low affinities for noradrenaline ( K m ~ 1100μ M) and adrenaline ( K m ~ 1700/gmM). This enzyme form is selectively inactivated by the monoamine oxidase type B inhibitor, I-deprenyl. In similar fashion to peripheral adrenergic neurons, PC12 cells share the capacity to express a tyramine releasable pool of catecholamines, a property entirely lacking in mature cultured chromaffin cells, even though the latter cells are capable of taking up tyramine by a cocaine sensitive process. However, both PC12 cells and chromaffin cells have a voltage and calcium dependent, K + induced, catecholamine release mechanism. Although previous studies have demonstrated that PC12 cells share some functional and biochemical properties with adrenergic neurons as well as chromaffin cells, the present study indicates that they have a closer resemblance to adrenergic neurons with regard to monoamine oxidase activity and tyramine induced catecholamine release than to chromaffin cells.

Enhancement of Ca2+-dependent high K+-evoked catecholamine secretion was observed after pretreatment of cultured bovine adrenal chromaffin cells with the phorbol ester 4B-phorbol 12-myristate 13-acetate (TPA) in the absence of added extracellular Ca2+. This effect of TPA was not reproduced when the secretagogues acetylcholine, nicotine, or veratrine were substituted for high K+. The implications of these results are discussed in relation to the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.

Barium ions enter chromaffin cells via voltage-sensitive calcium channels, although the intracellular site of barium action is distinct from that of calcium. The entry of barium primarily through voltage-sensitive channels was indicated by experiments showing inhibition of 133Ba2+ uptake by blockers of voltage-dependent calcium channels. In addition, 133Ba2+ uptake was stimulated by 50 mM KCl but not by nicotine. Furthermore, 133Ba2+ uptake was inhibited by hyperosmolarity, which specifically blocks the voltage-sensitive calcium channel but not the receptor-associated calcium channel. These conclusions from studies on barium uptake were also borne out by experiments measuring catecholamine secretion. Thus, blockers of voltage-dependent calcium channels which inhibited barium uptake also inhibited barium-induced catecholamine secretion. In other experiments, simultaneous stimulation with nicotine and barium in the presence of calcium evoked coincident and additive catecholamine secretion. By contrast, when 50 mM KCl was substituted for nicotine in the same experimental design, barium ions inhibited potassium-induced catecholamine secretion at low calcium concentrations. Only at high calcium concentrations were barium-induced and potassium-induced secretion additive. These data also indicate that barium and calcium compete at the voltage-sensitive pathway. Furthermore, these additivity data suggest that once inside the cell, barium and calcium have two distinct mechanisms of action. As predicted by this hypothesis, in digitonin-permeabilized chromaffin cells either calcium or barium stimulated catecholamine release, and in the presence of both cations catecholamine secretion was equivalent to the sum of secretion with either cation alone. Additional support of this concept comes from experiments showing that while calcium-mediated catecholamine secretion is sensitive to trifluoperazine and imipramine, barium-mediated secretion is not. Taken together, all these data indicate that there are two distinct intracellular sites of action for barium and calcium. In contrast to catecholamine secretion, non-exocytotic ascorbic acid secretion was induced by nicotine and potassium in the presence of calcium, but not by barium alone. These data provide additional evidence that barium acts by a different mechanism than calcium, in still another secretory system in chromaffin cells.

Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d -Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.

Synthetic oligopeptide inhibitors of metalloendoprotease activity were found to inhibit catecholamine release from intact bovine adrenal chromaffin cells. The efficiency of these compounds in blocking secretion was dependent on the type and dose of the secretagogues employed. By contrast, catecholamine release from digitonin-permeabilized cells stimulated with micromolar calcium was virtually not affected. Using a different model system mimicking protein-mediated membrane fusion during exocytosis (Bental, M., Lelkes, P.I., Scholma, J., Hoekstra, D., and Wilschut, J. (1984) Biochim. Biophys. Acta 774, 296-300) we found that exposure of chromaffin granules to a genuine metalloendoprotease, thermolysin, impaired their fusion competence with liposomes. The same oligopeptide inhibitors of metalloendoprotease activity that interfered with secretion from the intact cells were also found to cause an increase in 45Ca2+ efflux concomitant with a slight elevation of the free intracellular calcium concentration [( Ca2+]i) to levels not sufficient to elicit secretion. Subsequent stimulation of the cells in the presence of the potent inhibitors resulted in a reduced increase in the cytosolic calcium concentration, as compared to nontreated control cells. The reduction in the secretagogue-evoked rise in [Ca2+]i was also dependent on the time of pretreatment of the cells with the metalloendoprotease inhibitors. Consistently, none of these effects were seen with structurally similar oligopeptides that are not metalloendoprotease substrates/inhibitors. We conclude that potent inhibitors of metalloendoprotease activity and hence, presumably, the enzymes per se modulate stimulus-secretion coupling by interfering with calcium homeostasis rather than directly with membrane fusion.

The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [3H]norepinephrine formation from [3H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent Km of the dopamine beta-hydroxylation system for external dopamine was approximately 20 microM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent Km and Vmax of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 microM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptake into chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine were unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents for hydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfers electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.

Cholinergic synaptosomes from squid brain were found to release almost 50% of their total endogenous ATP when exposed to veratridine, an alkaloid which activates action potential sodium channels in nervous tissue. Veratridine also depolarizes synaptosomes and induces transmitter release by a mechanism which is dependent upon free Ca++ in the medium and is inhibited by tetrodotoxin, a specific veratridine inhibitor. ATP release activated by veratridine was also found to be calcium dependent and tetrodotoxin-sensitive. A new filter assay was developed to measure the kinetics of ATP release quantitatively, and veratridine-activated ATP release from synaptosomes was found to be complete in less than 30 seconds. Since ATP is a major component of cholinergic vesicles, this finding supports the concept that transmitter release from synaptosomes may occur from a vesicular rather than from a cytoplasmic pool.

Adenylate cyclase, dopamine-β-hydroxylase and cytochrome b562 have been found to co-equilibrate on equilibrium sucrose gradients of lysed chromaffin granule membranes from bovine adrenal medulla. Peak activities for these enzymes, as well as maximum membrane protein concentration, were found to coincide at d = 1.11 gm cm−3, and the ratios of adenylate cyclase to both dopamine-β-hydroxylase and cytochrome b562 were constant across the entire peak. Adenylate cyclase activity has been reported previously to co-purify with chromaffin granule membranes, and we conclude, on the basis of these new data, that adenylate cyclase is also an intrinsic granule membrane enzyme.

Synaptosomes derived from squid optic lobes were found to have specific, high affinity uptake systems for a variety of neurotransmitters. Chlorpromazine (CPZ) inhibited the uptake of l -noradrenaline ( l -NA) and 5-hydroxytryptamine (5-HT) in a competitive fashion. ( K i = 1.7 × 10 −7 M and 2.5 × 10 −7 M , respectively.) By contrast, CPZ (10 −5 M ) did not affect uptake of choline, aspartate, glutamate or glycine. These results suggest that permeation mechanisms for both l -NA 5-HT may have similar CPZ-sensitive sites.

It has been proposed (Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866) that synexin, an adrenal medullary protein that causes Ca2+-dependent aggregation of isolated chromaffin granules, might be the intracellular receptor for Ca2+ in the process of exocytosis. We now report that Ca2+ interacts directly with isolated synexin, inducing rapid self-association. Mg2+ or Sr2+ cannot substitute for Ca2+, and Ba2+ is only weakly effective at stimulating self-association. We have analyzed 90 degree light scattering data to determine a titration curve for the activation of synexin by Ca2+ in the self-association reaction. The curve has a Hill coefficient of 2.3 and is half-maximal at 200 micron Ca2+, which correlates exactly with the Ca2+ titration curve for the aggregation of chromaffin granules by synexin. Electron microscopy of negatively stained samples reveals that synexin monomers associate to form 50 A by 150 A rods which in turn associate side to side and end to end to form bundles of parallel rods. We suggest that synexin exists as a soluble monomer in the cytoplasm of the resting chromaffin cell and that when the cell is stimulated to secrete, the increase in the intracellular concentrations of Ca2+ causes synexin to form rods, similar to those formed in vitro, which bind to chromaffin granule and plasma membranes, joining them together to form the "pentalaminar" fusion complexes characteristic of the first step in exocytosis.

Ascorbic acid was found to be secreted from cultured bovine adrenal chromaffin cells coincidentally with catecholamines using a variety of secretagogues, including veratridine, nicotine, acetylcholine, and potassium chloride. Secretion of ascorbic acid was measured by preloading the cells for 3 h with (R)-[14C]ascorbic acid and then quantitating release of the label. The newly transported ascorbic acid constituted nearly 90% of total cellular ascorbic acid. Although chromaffin granules are known to contain endogenous ascorbic acid, only 16% of the total labeled ascorbic acid taken up by cultured cells was localized to the chromaffin granule fraction. Under the same isolation conditions, 95% of the endogenous catecholamines was found in the granule fraction. The secretion of both (R)-[14C] ascorbic acid and catecholamines was calcium dependent. However, only catecholamine secretion was effectively inhibited by the presence in the medium of isethionate, an impermeant ion that inhibits catecholamine secretion from isolated chromaffin granules. Analysis of the ratios of cellular and granular (R)-[14C] ascorbate as well as secreted (R)-[14C]ascorbate and catecholamines revealed that the secreted (R)-[14C]ascorbic acid did not come exclusively from the chromaffin granule fraction but also from a nonsedimenting cytoplasmic compartment. Indeed, for all four agonists, the amounts of (R)-[14C]ascorbic acid secreted were more than 1.5 times greater than could be accounted for by chromaffin granule ascorbate alone. We took these data to indicate that, while ascorbic acid and catecholamines were secreted concomitantly, the sources of the secreted substances were not necessarily identical.

ATP-stimulated release of epinephrine and protein from isolated chromaffin granules of the bovine adrenal medulla has been characterized with respect to pH optimum, substrate requirements, and temperature. Chromaffin granules incubated at 37 °C under optimal conditions released virtually all of their epinephrine and soluble protein within 10 min. ATP-stimulated epinephrine release was optimal at pH 5.8–6.2 and was five times greater than at pH 7.0–7.4. Magnesium and chloride were absolutely required for this process. Magnesium stimulated release at concentrations up to 1.0 m m ; however, it was moderately inhibitory at higher concentrations. The dependence of release on Cl − exhibited positive cooperativity and was nonsaturable. At 90 m m Cl − and 1.0 m m Mg 2+ , ATP-stimulated epinephrine release followed Michaelis-Menten kinetics with a K 1 2 of 0.2 m m . The release of soluble chromaffin granule proteins closely paralleled epinephrine release under all conditions tested, while membrane components were not released. Analogs of ATP substituted at the β-γ position with methylene or imino groups were also capable of stimulating release from granules. These ATP analogs had reduced affinity and lower activity than ATP itself. ATP analogs substituted at the α-β position were essentially inactive but were potent inhibitors of ATP-stimulated release. We conclude that the regulation of release from granules by ATP is rapid and specific and may not depend on hydrolysis of ATP at the β-γ position. This ATP-dependent reaction may be involved in the cellular process of exocytosis.

Summary A three-dimensional iso-density map of negatively stained low density lipoprotein (LDL) from human serum has been constructed. Iso-density data were collected from electron micrographs of LDL by scanning at one-micron intervals with a Tech/Ops Isodensitracer. Isodensity contour data were plotted directly on X-Y coordinates. A direct 50-fold enlargement of the particle was achieved with a significant reduction in the signal to noise ratio when compared to conventional enlargement methods. The best three-dimensional distribution of the isodensity data was that of a pentagonal-dodecahedron with isodensity maxima at the vertices. Based on this and other studies (1) these maxima were thought to represent protein subunits.

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The Keq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S20,w of 8, while the monomer had an S20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.

The chromatographic behavior of β-galactosidase from the constitutive strain 3300 of Escherichia coli, K-12, was studied with derivatives of agarose and polyacrylamide which contained the substrate analogue inhibitor, p-aminophenyl-β-dthiogalactopyranoside, covalently attached in various ways. A two-step purification of this enzyme from extracts could be achieved with selective agarose adsorbents provided the ligand was placed at a sufficient distance (about 21 A) from the matrix backbone by interposing a long hydrocarbon "arm," 3-aminosuccinyl-3'-aminodipropylamine. The various polyacrylamide-ligand derivatives studied were ineffective in adsorbing the enzyme. The enzyme adsorbed tightly at neutral pH to columns containing the substituted resin, and elution was achieved with buffers having a pH of 10. Buffers containing high concentration (0.05 m) of the substrates, lactose, isopropyl-β-d-galactopyranoside, or o-nitrophenyl-β-d-galactopyranoside, did not affect elution of the enzyme and were rapidly hydrolyzed by the enzyme adsorbed to the column. The enzyme obtained by these procedures was pure by disc gel electrophoresis and it possessed the physical and chemical characteristics of the protein purified by conventional procedures. The specific activity was between 300,000 and 320,000 units per mg. The procedure can be carried out on a preparative scale, and the adsorbent may be used several times without appreciable impairment of the binding capacity. The enzyme remains active while bound to the column and it can be readsorbed and eluted without apparent deleterious effects. The monomeric form of the enzyme also binds strongly to the substituted agarose, and it can be eluted together with the active tetramer species with sodium borate, pH 10. The ability of the enzymatically inactive monomer forms to bind to the ligand column suggests that this affinity chromatographic method may be used in the purification of catalytically inactive mutant forms which retain the ability to bind substrate analogues.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.