Search

A novel class of 1H-(benzimidazol-2-yl)-1H-pyridin-2-one inhibitors of insulin-like growth factor I (IGF-1R) kinase is described. This report discusses the SAR of 4-(2-hydroxy-2-phenylethylamino)-substituted pyridones with improved IGF-1R potency.

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595–605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in α1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti–PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2(cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2phosphorylation was lengthened to 2from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2translocated to the membrane in the presence of extracellular Ca2+went from 95% for spread cells. In the absence of Ca2+only 55–65% of the total cPLA2was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2from cytosol to membrane was Ca2+dependent, phosphorylation of cPLA2was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2phosphorylation and the release of AA.

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKC epsilon, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Immunization of the autoimmune mouse strain (M × A) Id/lpr with Ars-KLH, has been shown to elicit a prolonged anti-Ars IdCR response similar to that found in A/J mice. Cell fusion of splenocytes from a diseased mouse previously immunized with Ars-KLH resulted in a monoclonal antibody, 1–52.30, that was found to express the strain A major cross-reactive idiotype, but failed to bind Ars. Nucleotide sequence analysis demonstrated that 1–52.30: (a) used the “canonical” combination of gene segments associated with this idiotype, and (b) exhibited a pattern of somatic mutation consistent with selection for high affinity Ars binding. Two amino acids, VL 91 and 93, were mutated in 36–65, the germline equivalent of the IdCR antibodies, to 1–52.30-like residues (91G → D, 93T → M). The results of the mutagenesis showed that changing a single light chain residue, VL 91, from glycine to aspartic acid, resulted in a dramatic loss of Ars binding activity.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. The three-dimensional structure of this ferredoxin has now been determined and refined to a crystallographic R value of 16.7%, with all measured X-ray data from 30.0 to 1.7 A. The molecular motif of this ferredoxin is similar to that of other plant-type ferredoxins with the iron-sulfur cluster located toward the outer edge of the molecule and the irons tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteinyl residues. The overall secondary structure of the molecule consists of seven strands of beta-pleated sheet, two alpha-helices, and seven type I turns. It is of special interest that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different in the heterocyst form of the protein. Three of these positions are located in the metal-cluster binding loop.

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.

The solution properties of two fluorescent lipophilic analogues were examined in conjunction with their ability to penetrate the tight junctions of bovine aortic endothelial cell monolayers. 5-(N-dodecanoyl)aminofluoroscein was shown to label both the apical and basolateral plasma membrane domains of confluent monolayers at 4 degrees C and pH 7.3, but 5-(N-hexadecanoyl)aminofluoroscein was shown to label only the apical membrane domain. When used under more soluble conditions at 20 degrees C and pH 8.5, both probes labeled apical and basolateral plasma membrane domains more equally. This indicates that solubility conditions, and not tight junctions, dictate the penetration of 5-(N-hexadecanoyl)aminofluoroscein from the apical to the basolateral plasma membrane domain.

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure of the complex formed between bovine beta-trypsin and the highly potent synthetic inhibitor 2-{3'-[5'-methoxy-2'-(N-p-diaminomethylphenyl)amido]-1'-pyrido}-5-(N-2''-t-butylethanol)amidobenzoic acid (C(28)H(32)N(5)O(6)) has been determined at 0.97 A resolution. X-ray intensity data were collected to 0.97 A under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R(cryst) factors of 13.4 and 12.6% and R(free) factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F(o) - F(c)| difference map, which accounts for an estimated 35% of all H atoms at the 2.5sigma level. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195-His57-Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N(delta1) and Asp102 O(delta1).

Transparent substrates having heterogeneities ranging from nanometer to micrometer lateral length scale were fabricated to study cell migration. The surfaces were generated using thin films of block copolymers and homopolymer blends on ultra smooth transparent polyethylene terephthalate films. Results show that the lateral size scale of the surface heterogeneities affects fibroblast (NIH-3T3) adhesion, spreading and motility. More specifically, fibroblasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology on the micron patterned surfaces resemble cells cultured in a 3D environment. These surfaces, therefore, can potentially be utilized as models to study cell behavior in physiologically relevant conditions which can add to our fundamental understanding of cell-substrate interactions and facilitate development of surfaces for medical devices.

Identification of endothelial cell–surface proteins as targets for diagnosis and treatment of disease

Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 μm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 462–469, 2004

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B4 (LTB4) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB4 signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB4. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE2 reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro.

A series of nonguanidine N1-activated C4-carboxy azetidinone tryptase inhibitors was prepared by solid-phase methodology to quickly assess the SAR associated with distal functionality on the N1-activating group. From these studies, potent inhibitors with improved specificity were discovered.

Adhesion of cells to an extracellular matrix (ECM) is involved in a number of biological phenomena including wound healing, embryogenesis, blood coagulation, and metastasis’. Cell-substrate adhesion has four phenotypically distinct and sequential stages: attachment, spreading, migration, and/or immobilization. We previously demonstrated that during integrin-mediated attachment of HeLa cells to a substrate made of collagen or fibroblasts to fibronectin, integrin receptors were clustered by the substrate leading to the phosphorylation and translocation of cytosolic phospholipase A2 (cPLA2) to the membrane to catalyze the release of arachidonic acid (AA)2-4. The liberated AA serves as a substrate for lipoxygenases (LOs) leading to the formation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). This results in the production of diacylglycerol (DAG) to activate protein kinase (PKC) by translocation to the membranes. The result of PKC activation is polymerization of actin to facilitate cell spreading’.

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.

Inhibition of PLA2 activity and rescue by addition of exogenous AA was used to demonstrate that AA production is essential for integrin-mediated NIH-3T3 murine cell spreading. Both AA release and cell spreading after attachment to a FN substrate were inhibited by the PLA2 inhibitor mepacrine. AA release was essential for signaling spreading since the inhibition of spreading induced by mepacrine was overcome by exogenous AA. Cells ectopically expressing full-length chicken beta1-integrins both released AA and spread fully on a substrate of anti-chicken beta1-integrin monoclonal antibody, and inhibition of PLA2 by mepacrine suppressed both spreading and AA release. Exogenous AA also reversed this mepacrine-induced inhibition of spreading. The role of the beta1-integrin cytosolic domain in AA release was examined by comparing responses of cells expressing full-length chicken beta1-integrins versus cells expressing a deletion mutant chicken beta1-integrin with a truncated cytosolic domain. Cells expressing a truncated chicken beta1-integrin released significantly less AA and failed to spread on the anti-chicken beta1-integrin antibody substrate. Furthermore, clustering full-length receptors with soluble antibody stimulated greater AA release than clustering of receptors having truncated cytosolic domains. These data suggest the beta1-integrin cytosolic domain is required for optimal PLA2 activation to produce AA necessary for cell spreading.

The regulated initiation of programs leading to cell death is responsible, at least in part, for maintaining homeostasis within the immune system. Two general mechanisms for the production of cytotoxicity in susceptible targets by T effector cells have been described, one involving Ca++ dependent, perforin/granzyme exocytosis, and the other involving Fas antigen (CD95) engagement1. Fas functions in target cells as a receptor that signals for apoptosis, and Fas has been implicated in the process of reducing lymphocyte numbers following acute immune responses, termed activation induced cell death2–8. Although classical CD8+ cytotoxic T lymphocytes have recently been shown to express both types of cytotoxic activity, a distinct class of T cells, with the phenotype of CD4+ Th1 cells, mediates target cell death primarily in a Fas-dependent fashion9–14.

The three-dimensional structure of the fatty-acid-binding protein isolated from the flight muscle of the desert locust Schistocerca gregaria has been solved and refined to a crystallographic R-value of 18.5% for all measured X-ray data from 30.0- to 2.2-A resolution. Crystals employed in the investigation were grown from 2.6 to 2.8 M ammonium sulfate solutions, buffered at pH 7.5 and containing 2-5% 2-methyl- 2,4-pentanediol. They belonged to the space group P2, with unit cell dimensions of a = 61.6 A, b = 44.8 A, c = 63.9 A, and /3 = 113.6O and two molecules per asymmetric unit. The protein fold consists of ten strands of antiparallel P-pleated sheet that wrap around to form a B-barrel. In addition, there are two small a-helices and six type I, two type 11, and two type 11' turns. The two molecules pack in the asymmetric unit as a dimer with a local 2-fold rotational axis. The subunit-subunit interface involves amino acid side chains located in the area of the helix-turn-helix motif and the turn between &strands E and F. It is this area that has been speculated to form the portal through which fatty acids enter the binding cavity. There are 23 solvent molecules that are conserved between the two independent molecules in the asymmetric unit. Nine of these waters play important structural roles. A three-dimensional comparison between the insect and human muscle fatty-acid-binding proteins shows that their a-carbons superimpose with a root-mean- square deviation of 0.77 A for 89 structurally equivalent atoms. This high tertiary homology between these two proteins is surprising in light of the fact that they developed independently for more than 500 million years.

The 127 major polypeptides obtained from the purified plasma membrane of Dictyostelium discoideum were examined using two-dimensional gel electrophoresis and a microcomputer-based videodensitometer. Plasma membrane proteins were analyzed at four discrete stages of concanavalin A induced cell surface capping; (i) the cell surface in the absence of ligand (unbound), (ii) the surface immediately after ligand binding (bound), (iii) the cell surface after receptors had patched (patched) and (iv) the cell surface after receptors had capped (capped). Plasma membranes were obtained at various stages of capping by using a colloidal silica density perturbation technique which immediately immobilized the proteins, preserving their lateral distribution in the bilayer during the isolation. Proteins were characterized with respect to post-translational modification changes resulting from the capping process as well as changes in their association with the plasma membrane fraction. Posttranslational changes of plasma membrane proteins, such as phosphorylation, methylation and proteolytic cleavage, were not observed during the four stages of capping. Myosin heavy chain phosphorylation, however, decreased almost twofold during patching and capping. Actin, which is known to colocalize directly underneath capped receptors did not appear to be recruited to the cap from the cytoplasm.

Plasma membrane proteins of the cellular slime moldDictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling withN-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphoryalation state, glycosylation state, copy number, isoelectric point, and molecular weight.

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to α-methylmannoside in concanavalin A-carbohydrate competition studies.

We have determined the effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the sulfate-binding protein. The equilibrium dissociation constants of the binding of sulfate ( K d = 0.12 μm ) and selenate ( K d = 5 μm ) were found to be pH independent over the range pH 5 to pH 8.1, whereas chromate binding exhibited a pH dependence that is approximately attributable ot the p K 2 of the chromic acid. Phosphate was bound with an affinity five orders of magnitude weaker than that of sulfate. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, we conclude that the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the sulfate-binding site is stringently designed to bind tightly tetrahedral, fully ionized, oxyacid dianions. The presence of a donatable proton on the ligand reduces binding energy by approximately 7 kcal/mol.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age-associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor-mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose-dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age-dependent increase in resistance to ionomycin-induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin-calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.

A fast and efficient procedure for the purification of plasma membranes from Saccharomyces cerevisiae is described. Protoplasts served as starting material. They were coated with cationic silica microbeads. After lysis, the plasma membranes were washed free from debris and cell organelles. This procedure resulted in a high yield (about 85%) of plasma membranes, as judged by measuring vanadate-sensitive ATPase as a plasma membrane marker. The enzyme was enriched 12-fold relative to the homogenate after lysis. Its specific activity was 1.5–2.0 μmol/min per mg protein, the pH optimum was 6.5, and 10 μM vanadate was sufficient to obtain maximum inhibition. Based on the assay of internal markers and electron microscopic studies, we found our preparation essentially free of contamination from other cell organelles.

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.

A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S3 cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally.

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells, grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins absorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.

A quantitative colorimetric assay for carbodiimides was developed. It was based on the formation of a chromophore involving the carbodiimide, pyridine:HCl, and a diamine. The diamine used for water-soluble carbodiimides was 1,2-diaminoethane and that for the non-aqueous carbodiimides was piperazine. The maximum absorbance in the visible spectrum for the chromophore was 400 nm for carbodiimides in the aqueous assay and 420 nm for water insoluble carbodiimides assayed in dimethylformamide. The optimum pH for both assays was 7.0 and the absorbance of the chromophore was linearly dependent with the concentration of carbodiimide. Concentrations as low as 50 μ m were quantitated. The assay was specific for the carbodiimide and not for intermediates or products formed when the carbodiimide was used in coupling reactions to form amide bonds.

Four proteins of molecular mass 102, 87, 45, and 38 kDa were isolated from plasma membrane preparations by affinity chromatography. The 102-, 87-, and 38-kDa proteins were shown to be collagen receptors involved in the adhesion of HeLa cells to a gelatin substratum. All four proteins were eluted by high salt from affinity columns made of either types I or IV collagen or type I gelatin. Generally, a total of six major proteins were found in the high salt eluates, although the relative amounts of each varied among experiments. Immunoprecipitation, immunoblotting, and limited peptide mapping indicated that the 102-kDa protein was most sensitive to proteolysis leading to the formation of proteins of molecular mass 58 and 54 kDa. Even in the presence of a mixture of protease inhibitors the 58-kDa fragment was usually the more abundant species. Lectin binding indicated that the 102-, 87-, and 38-kDa proteins contain carbohydrate. Phase-partitioning with Triton X-114 and the need to solubilize the proteins in Triton X-100 indicated that the 102-, 87-, 45-, and 38-kDa proteins have a hydrophobic domain. The 87-kDa protein partitioned exclusively with the detergent-rich phase, suggesting that it is the most hydrophobic. Cell surface labeling with 125I indicated that the four proteins have an extracellular domain. Four criteria were used to determine which of the four proteins are collagen receptors mediating cell-substrate adhesion: 1) during HeLa cell adhesion, proteins with Mr values similar to all four proteins or their peptide fragments were cross-linked to a gelatin substratum derivatized with a photoactivatable probe; 2) a pentapeptide containing the Arg-Gly-Asp cell recognition sequence eluted the same four proteins as those found by high salt elution of collagen affinity columns; 3) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, inhibited the spreading of HeLa cells on a gelatin substratum; 4) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, bound to culture dishes substituted for gelatin in mediating the spreading of HeLa cells. Taken together, the data suggest that the 102-, 87-, and 38-kDa proteins are collagen receptors involved in HeLa cell adhesion. Although the 45-kDa protein has two of the characteristics of a collagen receptor defined here, it does not fit the criteria for one involved in cell-substratum adhesion.

A disrupted spinach chloroplast preparation readily synthesized [ 14 C]α-linolenate from [2-14C]acetate under anaerobic conditions. It can be shown by degradation data that [14C]oleate is not a precursor of [14C]linolenate and that cis 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [14C]linolenate.

An enzyme system present in the stromal fraction of spinach chloroplasts specifically elongated medium chain trienoic fatty acids to α-linolenic acid with either acetyl or malonyl CoA. Medium chain and long chain saturated and monounsaturated fatty acids as well as α-linolenic acid were ineffective substrates for elongation. These results fulfill an essential requirement for the proposed two pathway systems for polyunsaturation in plants.

Carbon dioxide from untreated and alloxanized rats was collected free of atmospheric CO2 and then analyzed for its stable carbon isotope (13C12C) ratio. Alloxan induced diabetic rats expired CO2 with a 13C12C ratio less than that for CO2 collected from untreated rats. The difference in 13C12C ratios indicated that alloxanized rats oxidized more lipid than control rats since rat lipid has a 13C12C ratio less than that of glycogen.

Chloroplasts isolated from immature leaves of spinach (Spinacia oleracea) differ in enzyme levels from those isolated from mature leaves. On a chlorophyll basis, immature chloroplast preparations had 5- to 6-fold higher capacity to synthesize fatty acids from 2-14C-acetate compared to plastids isolated from mature leaves. This difference was correlated with higher activities for the enzymes, acetyl coenzyme A synthetase, malonyl coenzyme A synthetase, acetyl coenzyme A carboxylase, and oleyl coenzyme A transferase in plastid pressates obtained from immature leaves. Disrupted chloroplast preparations from both mature and immature leaves retained the ability to incorporate 2-14C-acetate into fatty acids in a pattern similar to that by isolated chloroplasts. 2-14C-Acetate, 2-14C-acetyl coenzyme A, 2-14C-malonate, and 1,3-14C malonyl coenzyme A were readily incorporated into a number of fatty acids. Moreover, the synthesis of oleate by chloroplast pressates from these substrates was strongly inhibited by KCN, flavin adenine mononucleotides and dinucleotides, and anaerobic conditions, while linolenic acid synthesis was unaffected by these compounds.

Acetazolamide (2-acetylamino-1,3,4-thiadiazole-5-sulfonamide), a compound commonly used to inhibit carbonic anhydrase has been ound to inhibit the photoreduction of 3-phosphoglyceric acid, NADP, and methyl viologen by isolated spinach chloroplasts. Acetazolamide had no effect on the photoreduction of methyl viologen by chloroplasts using ascorbate plus 2,6-dichloroindophenol, as an electron donor, indicating the site of the acetazolamide inhibition was not in photosystem I. There was no acetazolamide effect on NADP reduction by isolated chloroplasts using hydroxylamine instead of water as the electron donor. The results indicate that the site of acetazolamide inhibition is near the water-splitting side of photosystem II.

The permeability of chloroplast membranes to acetate was studied by measuring the direct movement of radioactive acetate into the organelle. The half-time for equilibration of 1 m m acetate across the envelope membrane was less than 5 sec while that for the lamellar membranes was greater than 30 min at pH 7.9. Evidence suggested that the undissociated acid penetrated faster than the acetate anion. Acetate was bound by the chloroplasts with an optimum pH of 6.5. The binding was enhanced fourfold by Mn2+ and Mg2+ with a reversal of the enhancement by EDTA. Binding was weak and saturated at 15–30 nmoles acetate/mg chlorophyll. It appeared that acetate was bound in the vicinity of the lamellar membranes. Permeation of acetate was much faster than and without effect on that of glutamate.

Isotope discrimination is a common feature of biosynthesis in nature, with the result that different classes of carbon compounds frequently display different (13)C/(12)C ratios. The (13)C/(12)C ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory CO(2) from potato discs in successive periods through 24 hr from the time of cutting-an interval in which the respiration rate rises 3-5-fold. The (13)C/(12)C ratio of the evolved CO(2) was determined for each period, and compared with the (13)C/(12)C ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the CO(2) matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each juncture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism-possibly the alpha-oxidation of long chain fatty acids-while the developed repiration represents conventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO(2) may be obscured by fractionation attending the refixation of CO(2) during respiration, and by CO(2) arising from dissolved CO(2) and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls.

Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds.

Glucoamylase (1,4-α- d -glucan glucohydrolase, EC 3.2.1.3) from A. niger was immobilized on cationic nonporous glass beads (13–44 μm) by electrostatic adsorption followed by rosslinking with glutaraldehyde. Over 80% of the enzyme's total soluble activity was expressed upon immobilization. d -Glucose production from maltodextrins was virtually complete, suggesting that the lack of pores can eliminate the problem of product reversion. Immobilized glucoamylase showed decreased stability upon heating, compared with the soluble enzyme .

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.

Solid glass beads for use in isolating cell membranes were coated with a stable, covalently attached layer of polylysine. The optimal conditions for coating the bead surface were established and the beads were tested by measuring the attachment of human erythrocyte plasma membranes. When compared to other beads, such as those with absorbed polylysine or protamine, none retained red-cell membranes as well as glass beads with covalently linked polylysine.

Erythrocytes were ionically attached to polylysine-coated beads 30 micrometers in diameter. The binding of the cells was so tenacious that lysis or disruption of the attached cells left the beads covered by plasma membranes whose cytoplasmic surface was exposed and accessible for further analysis.