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Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m7GMP) or diphosphate (m7GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5′ nucleotidase from Drosophila that cleaves m7GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m7GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5′ nucleotidase activity with a high affinity for m7GMP. CMP is a slightly better substrate but again with a higher Km. The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m7GMP as a substrate. We suggest that the m7G-specific nucleotidases protect cells against undesired salvage of m7GMP and its incorporation into nucleic acids. Background: mRNA decay releases, in addition to the regular nucleotides, 7-methyl GMP derived from the 5′ cap. Results: We describe new members of the 5′ nucleotidase family degrading 7-methyl GMP to 7-methylguanosine and orthophosphate. Conclusion: Cells have mechanisms to prevent potential salvage of 7-methyl GMP. Significance: 7-Methyl GMP degradation may be important to prevent its incorporation into nucleic acids.

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.

Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile170, Ser172, and Ser226 with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys30, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

Bisphosphate 3′-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3′-nucleotidase and its substrate, 3′-phosphoadenosine 5′-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3′-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3′-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3′-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0–8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn2+ > Co2+ > Ni2+ > Mg2+; YfbR: Co2+ > Mn2+ > Cu2+; YjjG: Mg2+ > Mn2+ > Co2+). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5′-monophosphates and ribonucleoside 3′-monophosphates with highest affinity to 3′-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P20–25). YfbR was strictly specific to deoxyribonucleoside 5′-monophosphates, whereas YjjG showed narrow specificity to 5′-dTMP, 5′-dUMP, and 5′-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.

Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3'-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3'-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys(3)-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by approximately 73 degrees upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in the Arabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3'-phosphoesterase acts as a "nick sensor" for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex.

SDT1 (suppressor ofdisruption of TFIIS 1, YGL224c, also known as SSM1, suppressor ofS-II null mutant 1) isSaccharomyces cerevisiae gene identified as a multicopy suppressor of 6-azauracil sensitivity in a null mutant of the transcription elongation factor S-II. We found that overproduction ofSDT1 caused hyposensitivity to not only 6-azauracil but also 5-fluorouracil and 5-fluorocytosine. This hyposensitivity was limited to pyrimidine derivatives, and no effect was observed for non-pyrimidine drugs including such clinically used anti-fungal drugs as amphotericin B and fluconazole. Purified recombinant SDT1 protein specifically dephosphorylated 5′-UMP and 5′-CMP. These results suggested that SDT1 conferred pyrimidine-specific hyposensitivity by dephosphorylating active metabolites of 6- or 5-modified pyrimidines, i.e. 6- or 5-modified UMP. This is the first description of a highly specific pyrimidine 5′-nucleotidase in S. cerevisiae.

Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5′-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2′-deoxyadenosine, with a 49-fold increase in the IC50 in HEK 293 and a greater than 400-fold increase in the IC50 in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC50 to 2′,3′-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC50 to 2′,3′-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs.

We describe the cloning and characterization of a novel bone-specific cDNA predicted to encode an extracellular matrix protein. This cDNA was identified by subtractive hybridization based upon its high expression in bone marrow-derived osteoblasts. By Northern blot analysis, we detected a single 2-kilobase mRNA transcript in bone, whereas no expression was detected in other tissues. Immunohistochemistry revealed that the protein was expressed highly in osteocytes within trabecular and cortical bone. RNA and protein expression analysis using in vivo marrow ablation as a model of bone remodeling demonstrated that this gene was expressed only in cells that were embedded within bone matrix in contrast to the earlier expression of known osteoblast markers. The cDNA was predicted to encode a serine/glycine-rich secreted peptide containing numerous potential phosphorylation sites and one RGD sequence motif. The interaction of RGD domain-containing peptides with integrins has been shown previously to regulate bone remodeling by promoting recruitment, attachment, and differentiation of osteoblasts and osteoclasts. Secretion of this RGD-containing protein from osteocytes has the potential to regulate cellular activities within the bone environment and thereby may impact bone homeostasis. We propose the name OF45 (osteoblast/osteocyte factor of 45 kDa) for this novel cDNA.

The 3'-nucleotidase/nuclease (3'-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3'-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an approximately 1000-fold up-expression of 3'-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3'-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the approximately 100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3'-NT/NU enzyme activity was mediated at the posttranscriptional level.

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.

The 5'(3')-nucleotidase earlier partially purified from rat liver by Fritzson and Smith [1971) Biochim. Biophys. Acta 235, 128-141) was purified 15,000-fold to apparent homogeneity from human placenta. The soluble enzyme is a homodimer with a native molecular mass of 44-45 kDa. It has a pH optimum between 6.0 and 6.5 and is absolutely dependent on Mg2+ ions. The enzyme dephosphorylates certain 2'-, 3'-, and 5'-nucleotides. Km values for 2'- and 3'-nucleotides are around 0.3 mM with no preference for either ribo- or deoxyribonucleotides. 5'-Deoxyribonucleotides are 10-fold better substrates than the corresponding ribonucleotides, with dIMP greater than dUMP greater than dGMP greater than dTMP. dAMP is a poor substrate, dCMP is essentially insert. In all cases the Km values are in the millimolar range. Of the different forms of nucleotidases characterized in animal cells, the 5'(3')-nucleotidase is unique in its preference for 5'-deoxyribonucleotides. In intact cells, a portion of de novo synthesized deoxyribonucleotides is degraded as part of a homeostatic mechanism regulating the size of deoxyribonucleotide pools. This requires the participation of one or several 5'-nucleotidases. The 5'(3')-nucleotidase may be one such enzyme.

To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse-labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form.

Plasma membranes were purified from bovine anterior pituitary tissue by differential and isopycnic centrifugation according to a modification of Neville's technique (Biophys. Biochem. Cytol. 8, 413, 1960). Plasma membranes, rough endoplasmic reticulum, and secretory granules sedimented at the interfaces of sucrose layers of different densities and were thus simultaneously obtained in high yield by this technique. The yields of these different fractions were, respectively, 1, 2.5, and 10 mg per g of original pituitary tissue. As shown by electron microscopy, the plasma membrane fraction was not contaminated by other identifiable subcellular components. Growth hormone and prolactin accounted for, respectively, 10 to 30% and 60 to 80% of the total proteins of secretory granules. There was a 7- to 14-fold enrichment of Mg2+-dependent-(Na+-K+)-stimulated ATPase and 5'-nucleotidase activities in the plasma membrane fraction relative to the total homogenate. Adenylate cyclase activity was approximately 10-fold higher in the plasma membrane fraction than in the original homogenate. NaF, 10 mm, gave a 4- to 10-fold stimulation of enzymatic activity in all fractions. Maximal activation of plasma membrane adenylate cyclase activity by Mg2+ and Mn2+ was observed, respectively, at concentrations of 3.5 and 2 mm. Ca2+ inhibited cyclic adenosine 3' : 5'-monophosphate formation at all concentrations studied.