Changes in cAMP-dependent protein kinase activity in the 10,000g sediment of sea urchin embryos during early development

cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.     

Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans.

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.     

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.     

Subcellular distribution and isoelectric heterogeneity of the substrate for ADP-ribosyl transferase from Clostridium botulinum

When the homogenate of bovine adrenal gland was subjected to subcellular fractionation, an Mr 21,000 substrate for botulinum ADP-ribosyl transferase was found not only in the membrane fractions but also in the cytosol; the amounts in the 10,000 x g precipitates and the 100,000 x g supernatant were about 21 and 56% of the total amount, respectively. Each fraction gave a single ADP-ribosylated protein band on SDS-polyacrylamide gel electrophoresis, but yielded on isoelectric focusing at least three bands between pH 5.5 and 6.0, suggesting the presence of multiple forms of the substrate of a similar molecular weight but different isoelectric points. ADP-ribosylated protein bands from the membrane and cytosol overlapped each other on both electrophoreses. After ammonium sulfate fractionation, the substrate from the cytosol showed requirement of divalent cations or guanine nucleotides for the reaction. Among cations tested, calcium, magnesium and manganese stimulated, whereas cadmium and lanthanum inhibited the reaction. Guanine nucleotides such as GTP, GDP and GTP-gamma-S also stimulated the substrate activity in the cytosol as that in the membrane fraction. However, no additive effects were observed when the nucleotides and cations were added together.     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C4 (LTC4) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added [3H] LTC4 by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted [3H] LTC4 mainly into [3H] LTE4 (83%) and, at a smaller extent, into [3H] LTD4 (4%). Intact [3H] LTC4 represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD4. In contrast, there was nearly no conversion of [3H] LTC4 (87% intact) in the presence of homogenized papilla. The metabolism of [3H] LTC4 by the glomeruli was time- and temperature-dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize [3H] LTC4. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform [3H] LTC4 into its metabolites, LTD4 and LTE4. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected [3H] LTC4 from its bioconversion. These results demonstrate that both glomeruli and papilla possess the gamma-glutamyl transpeptidase and dipeptidase necessary to process LTC4. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

Characterization of gill (Na + K)-activated adenosine triphosphatase from chinook salmon, Oncorhynchus tshawytscha.

(Na+K)-activated ATPase activity from gills of yearling spring chinook was examined using a new rapid assay method. Characterization of the enzyme activity was performed. Optimal activity was obtained at pH 7.2 in the presence of 240 mM NaCl, 120 mM KCl, 20 mM MgCl2 and 10 mM Na2ATP. Maximal inhibition of the enzyme was observed in the presence of 0.5 mM ouabain. Differential centrifugation indicated that 75% of the enzymatic activity was sedimented at 1000 x g. Only 8% of the activity was found in the microsomal pellet. Treatment with 0.1% sodium deoxycholate liberated activity from the 1000 x g pellet and elevated the activity. This treatment caused a loss of 20% of the original activity of the preparation. Statistical analysis of the sampling procedure for gill (Na+K)-activated ATPase activity indicated that there was small variation in the technique itself when compared to variation between the individual gill arches and between individual fish. Results indicate that for meaningful comparisons of groups of fish, the sampling of the gill arches must be standardized and a large number of individual fish must be sampled.     

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Biosynthesis of chitin by particulate fractions from Cunnighamella elegans.

The enzyme chitin synthetase (UDP-acetylaminodeoxyglucosyl transferase, EC 2.4.1.16) in Cunninghamella elegans has been investigated. The enzyme was present in the microsomal, cell wall, mitochondrial and the soluble cytoplasmic fraction of the mycelium, with the former having the highest specific activity. The properties of the enzyme in this fraction were investigated; the Km for UDP GlcNAc was 1.23 mM and 2.08 mM GlcNAc in the presence of 1 mM UDP GlcNAc. The temperature optimum was between 26 degrees and 29 degrees C and maximal activity was at pH 6.25. Mg++ ions had no effect on chitin synthesis, but soluble chitodextrins inhibited the enzyme. The production of chitin synthetase was correlated with the growth of the fungus, maximum activity being found during the late exponential phase of growth. Chitin was confirmed as the sole product of enzyme action, by digestion with chitinase.     

Inhibition and activation of mannan synthesis in Saccharomyces cerevisiae spheroplast lysates.

Mannan synthetase activity in spheroplast lysates prepared from Saccharomyces cerevisiae was measured by following the incorporation of [14C]mannose from guanosine 5'-diphosphate-[14C]mannose into material precipitable with cold 0.3 M perchloric acid. When enzyme activity was assayed at high concentrations of spheroplast lysate protein (10 mg/ml) in the presence of 7.5 mM MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the spheroplast lysate at 4 degrees C for 16 to 32 h before assay, by repeated freezing and thawing of the spheroplast lysate, or by the omission of MnCl2 from assay mixtures. The addition of ethylenediaminetetraacetic acid or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for spheroplast lysate as the source of mannan synthetase. The supernatant fluid obtained by centrifuging spheroplast lysate at 100,000 x g, when added to assay mixtures containing either spheroplast lysate preincubated at 4 degrees C or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 mM MnCl2 and was destroyed by heating the supernatant fluid at 60 degrees C for 10 min, or by trypsin treatment at 30 degrees C. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in spheroplast lysates may be modulated by preincubation at low temperature or by varying the available Mn2+ concentration.     

Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion

Human platelet soluble guanylate cyclase activity was studied with respect to the function of its heme-containing regulatory subunit. As an enzyme source, the 10,000 x g supernatant was used and, since its specific activity proved to be too low for inhibition studies, also a partially purified preparation was employed. The partially purified enzyme was stimulated about 2.5-fold by carbon monoxide and this effect was abolished by illumination with visible light. Sodium nitroprusside also increased the basal activity about fourfold, which, however, is much less than the greater than 100-fold stimulation seen with the supernatant. Superoxide anions generated by the xanthine/xanthine-oxidase system were strongly inhibitory in the enriched preparation as well as in the CO-stimulated platelet supernatant (median effector concentration = 0.1 mU/ml). Unlike CO and NO, the effect of superoxide cannot be mediated through the heme-containing regulatory subunit, since heme-free enzyme, which could not be activated by NO or CO, was inhibited to the same extent as the heme-containing enzyme. Superoxide dismutase did not influence the basal activity, but resulted in a synergistic stimulation in the presence of CO. When Mn2+ replaced Mg2+ as a cofactor, the basal activity was higher but superoxide could not inhibit the enzyme, possibly due to the superoxide-dismutase-like activity of Mn2+. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with endothelium-derived relaxing factor, recently identified as NO, could form a physiologically relevant regulatory effector system.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.     

Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally cryptorchid rat testis.

The effect of experimental cryptorchidism on the level of oxidative stress and antioxidant functions in rat testis was studied. Adult male Sprague-Dawley rats were rendered unilaterally cryptorchid (by suturing one testis to the abdominal wall) and killed 1, 3, or 7 days after the operation. As an indicator of oxidative stress, lipid peroxidation was measured by the diene conjugation method in testis homogenates. The activities of the antioxidant enzymes were determined either in the 10,000 x g supernatant fraction (glutathione [GSH] peroxidase, GSH transferase, hexose monophosphate shunt) or in crude testis homogenates (superoxide dismutase, catalase). An expected reduction (48%) in weight of the abdominal testes was evident by postoperative Day 7. The catalytic activities per testis of superoxide dismutase (Cu/Zn form) and catalase were found to decrease in cryptorchidism. The effect was seen on the first postoperative day and was most profound on Day 7 after surgery. The principal antioxidant enzyme, superoxide dismutase, was most sensitive to cryptorchidism, the activity in the abdominal testes being 74% or 85% (per gram of tissue or per whole testis, respectively; p less than 0.01). After impairment of the reactive oxygen detoxifying capacity, lipid peroxidation was increased in the abdominal testis by 46% (p less than 0.01) on postoperative Day 7. Slight concomitant increases were detected in the activities of GSH-peroxidase (p less than 0.01), GSH-transferase (p less than 0.001), and the hexose monophosphate shunt (p less than 0.001). This effect was seen only when calculated per gram of tissue, not per whole testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity determination, kinetic analyses and isoenzyme identification of gamma glutamyltransferase in human neutrophils

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) which hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH concentration. GGT is a membrane enzyme present in leukocytes and platelets. Its activity has also been observed in human neutrophils. In this study, GGT was purified from Triton X-100 solubilized neutrophils and its kinetic parameters were determined. For kinetic analyses of transpeptidation reaction, gamma-glutamyl p-nitroanilide was used as the substrate and glycylglycine as the acceptor. Apparent K(m) values were determined as 1.8 mM for gamma-glutamyl p-nitroanilide and 16.9 mM for glycylglycine. The optimum pH of GGT activity was 8.2 and the optimum temperature was 37 degrees C. It had thermal stability with 58 % relative activity at 56 degrees C for 30 min incubation. L-serine, in the presence of borate, was detected as the competitive inhibitor. Bromcresol green inhibited neutrophil GGT activity as a noncompetitive inhibitor. The neutrophils seem to contain only the isoenzyme that is present in platelets. We characterized the kinetic properties and compared the type of the isoenzyme of neutrophil GGT with platelet GGT via polyacrylamide gel electrophoresis (PAGE) under a standard set of conditions.     

Human liver alpha-L-fucosidase. Purification, characterization, and immunochemical studies.

Human liver alpha-L-fucosidase has been purified 6300-fold to apparent homogeneity with 66% yield by a two-step affinity chromatographic procedure utilizing agarose epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all six isoelectric forms of the enzyme were purified. Polyacrylamide gel electrophoresis of the purified alpha-L-fucosidase demonstrated the presence of six bands of protein which all contained fucosidase activity. The purified enzyme preparation was found to contain only trace amounts of seven glycosidases. Quantitative amino acid analysis was performed on the purified fucosidase. Preliminary carbohydrate analysis indicated that only about 1% of the molecule is carbohydrate. Gel filtration on Sepharose 4B indicated an approximate molecular weight for alpha-L-fucosidase of 175,000 +/- 18,000. High speed sedimentation equilibrium yielded a molecular weight of 230,000 +/- 10,000. Sodium dodecyl sulfate polyacrylamide gels indicated the presence of a single subunit of molecular weight, 50,100 +/- 2,500. The enzyme had a pH optimum of 4.6 with a suggested second optimum of 6.5. Apparent Michaelis constants and maximal velocities were determined on the purified enzyme with respect to the 4-methylumbelliferyl and the p-nitrophenyl substrates and were found to be 0.22 mM and 14.1 mumol/mg of protein/min and 0.43 mM and 19.6 mumol/mg of protein/min, respectively. Several salts had little or no effect on fucosidase activity although Ag+ and Hg2+ completely inactivated the enzyme. Antibodies made against the purified fucosidase were dound to be monospecific against crude human liver supernatant fluids and the pure antigen. No cross-reacting material was detected in the crude liver supernatant fluid from a patient who died with fucosidosis.     

Content and formation of prostaglandins and distribution of prostaglandin-related enzyme activities in the rat ocular system

The steady-state levels of prostaglandin D2, E2 and F2 alpha in the rat eye were 0.5, 0.1 and 1.0 ng/g, respectively, which increased differently among the prostaglandins after a 40-min incubation of the homogenate at 37 degrees C (to 23, 12 and 14 ng/g, respectively). When the eye was dissected into anterior uveal, scleral, and retinal complexes, prostaglandin D2 was formed in the highest degree in all the complexes, whereas prostaglandin E2 and F2 alpha formation was specific to given ocular regions. Three prostaglandin synthetase activities with similar Km values (20-40 microM) were found in the 10,000 X g supernatant of these tissues, i.e., GSH-independent and soluble D, GSH-dependent and membrane-bound E, and soluble F synthetase activities. These enzyme activities correlated well with the prostaglandin formation in each tissue. D synthetase activity being highest in all the tissues (11-25 nmol/min per g). Three types of prostaglandin-catabolizing enzyme activities were detected in the 100,000 X g supernatant of the tissues, i.e., type II 15-hydroxy dehydrogenase (Km = 10-30 microM), 9-keto (500 microM) and 11-keto reductase (2.5 mM). The activity of the dehydrogenase was low even in the retina, the tissue with the highest levels (0.51, 0.35 and 0.15 nmol/min per g for prostaglandin E2, F2 alpha and D2, respectively).     

Physiological Studies on Thiobacilli

Thiosulfate was oxidized to tetrathionate and an unknown oxidized sulfur compound. The activity was found in the particulate fraction (a precipitate obtained by centrifuging (at 90,000 × g for 60 min) the supernatant of centrifugation at 10,000 × g for 30 min). Treatment with deoxycholate caused inactivation of the enzyme. Considering from these points and electronmicrograph, the enzyme would be located on the cell membrane.     

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10(-5) M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10(-5) M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.