Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and Mg²⁺-(Na⁺ + K⁺)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na⁺ + K⁺)-ATPase was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). (Na⁺ + K⁺)-ATPase activity was undetectable in thymocytes. However, in spleen lymphocytes (Na⁺ + K⁺)-ATPase activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion

Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10⁻⁷ M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E₁ and 184% by prostaglandin E₂.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca²⁺ above 10⁻⁶ M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca²⁺.     

Glial tumor cells deficient in thymidine kinase: isolation and characterization

Rat astrocytoma cells were treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine and grown in the presence of high concentrations of the thymidine analog 5-bromo-2′-deoxyuridine. A surviving clone, designated C₆TK⁻, lacked thymidine kinase but exhibited differentiated functions such as catecholamine-sensitive adenylate cyclase activity, cortisol-inducible glycerol-1-phosphate dehydrogenase activity, and S-100 protein biosynthesis.     

Hormonal control of FAT cells adenylate cyclase

Summary Fat cells were preincubated for 2 h in the presence and absence of growth hormone (GH) and Dexamethasone (Dex) before the addition of increasing concentrations of either epinephrine, theophylline or glucagon and final incubation of the cells for an additional 5 minutes. GH and Dex increased by 85%, 28% and 72%, respectively, the cAMP levels reached in the sole presence of 10^–5 m epinephrine, 10^–2 m theophylline or 5 × 10^–5 m glucagon. An adenylate cyclase particulate preparation shows that epinephrine decreases Km from 2mm to 0.6mm and increases V_max and the strength of interaction value (n) from 0.91 to 1.75.     

The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5′-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg²⁺ or Mn²⁺. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5′-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.     

LM fibroblast plasma membrane subfractionation by affinity chromatography on Con A-sepharose

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-side-out orientation of the vesicles. Similarly the asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5′-nucleotidase activity, and (Na⁺ + K⁺)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and Arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.     

Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO₃ buffer or by exposure to the chelator ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5′-(β-γ-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca²⁺, Mg²⁺ or Mn²⁺. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca²⁺. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca²⁺ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P.Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca²⁺-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca²⁺. The effects of low concentrations (less than 100 μM) of the phenothiazine could be reversed totally by Ca²⁺ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca⁺ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.     

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca²⁺-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.     

Quantitation of epinephrine- and glucagon-sensitive adenylate cyclases of rat liver implications of alterations of enzymatic activities during preparation of particulate fractions and membranes

Stimulated and basal adenylate cyclase activities from livers of young and old rats were lower in particulates than in homogenates. Particulates were compared to homogenates by reconstituting the suspensions to the volume of the homogenates from which they were derived; enzyme activities in paired homogenates and particulates therefore reflected the same amounts of membrane-bound enzyme. The magnitude of the losses of hormone-sensitive activities in particulates was dependent on the age and sex of the animals and the concentrations of hormone. Particulates from 3-month-old animals showed glucagon-( (1 · 10^?5 M) and epinephrine-sensitive (1 · 10^?4 M) activities which were 67 and 78% of homogenate activities, respectively; particulates from 24-month-old animals had activities relative to homogenates of 55% for glucagon and as low as 32% for epinephrine. The glucagon dose vs. response curve in particulates and membranes showed maximal activity at 1 · 10^?7 M glucagon while in homogenates activity increased linearly with increasing glucagon concentrations up to 1 · 10^?5 M. Losses of basal and anion-stimulated activities were similar at both ages. Fluoride and azide stimulations relative to basal activities were greater in particulates than in homogenates, while relative epinephrine activity was lower in particulates, suggesting qualitative alteration of adenylate cyclase during preparation of particulates. These studies show that adenylate cyclase activity in rat liver is presently best quantitated in homogenates and suggest caution in comparisons of enzyme activities based on particulates or membranes prepared from animals of differing physiologic states.     

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs

Summary Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon_1–21 and glucagon_1–6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon_2–29 ([1-des-histidine]-glucagon), [1-N-carbamoylhistidine]-glucagon and [1-N-carbamoylhistidine, 12-N-carbamoyllysine]-glucagon are all partial agonists.These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-N-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA₂ values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals.Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.     

Desensitization of adenylate cyclase and cyclic AMP flux during the early stages of liver regeneration

Liver regeneration is controlled by a complex network of interactions between hormones, growth factors, and a variety of hepatotrophic factors. Transient increases in cAMP in the early stages of liver regeneration that are necessary for DNA synthesis and subsequent mitosis have been reported; however, studies on the mechanisms that control cellular cAMP levels during liver regeneration, namely adenylate cyclase activity, cAMP-dependent phosphodiesterase activity, and cAMP efflux from the cell, have been generally incomplete. In this study we have shown that although there are three peaks in intracellular cAMP levels in the first 24 hours after partial hepatectomy, the adenylate cyclase activity stimulated by glucagon, prostaglandin E2, adrenaline, and fluoride in vitro decreases with time. KD and BMAX of hepatocyte glucagon and beta receptors were similar to the sham controls. Our results are consistent with a mixed homologous/heterologous desensitization of the adenylate cyclase system. There was also a loss of cAMP-dependent phosphodiesterase activity after partial hepatectomy. We speculate that even though the hormone-stimulated adenylate cyclase system has been desensitized, the system retains the ability to respond to the transient pulses of the variety of hormones secreted after partial hepatectomy and thus raise the intracellular concentration of cAMP. The decrease in cAMP-dependent phosphodiesterase may be necessary to prevent rapid breakdown of cAMP.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques.In the closed-loops, 5 mM deoxycholate selectively inactivated (Na⁺ + K⁺)-ATPase, and net secretion of Na⁺ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-O-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5±2.9 % per g wet wt of gut) greatly exceeded those of taurocholate (4.3±1.0).In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na⁺ and Cl⁻, and inhibited active glucose transport; concomitantly “total” ATPase, (Na⁺ + K⁺)-ATPase, and Mg²⁺-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg²⁺-ATPase activity. Taurocholate and cholate at 1 mM had no effect on transport or (Na⁺ + K⁺)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate.These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na⁺ + K⁺)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function

A novel mutant of the LLC-PK₁ renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V₂ receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK₁ cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5′-[β,γ-imino] triphosphate, 8-bromo adenosine-3′,5′-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK₁ and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V₂ receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V₂ receptor gene using DNA-transfer.     

Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa

Rat gastric mucosa was shown to contain a Mg²⁺-dependent ATPase which is stimulated by HCO₃⁻ at pH 8–9.Triton X-100 solubilizes this HCO₃⁻-stimulated, Mg²⁺-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg²⁺-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO₃⁻ addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg²⁺-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO₃⁻. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO₃⁻; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO₃⁻.The bicarbonate-stimulated fraction of the Mg²⁺-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO₃⁻ concentration. In addition, SCN⁻ is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO₃⁻-stimulated ATPase in the gastric secretion of H⁺ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H⁺ secretion, attention can be directed towards the Mg²⁺-dependent ATPase found to be associated with microsomes.     

Solubilization of rat heart sarcolemma 5′-nucleotidase by phosphatidylinositol-specific phospholipase C

The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5′-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5′-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same K_m for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in K_m for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5′-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.     

Adenylate cyclase and cyclic nucleotide phosphodiesterases in the developing rat liver

A potential regulatory role for the cyclic nucleotides during liver morphogenesis will be better understood as the development of various components of the cyclic nucleotide system are characterized. Accordingly, adenylate cyclase response to glucagon and 5′-guanylimidodiphosphate (Gpp(NH)p) and the specific activities, cellular distributions, and kinetic constants (V and K_m) of the cyclic AMP and cyclic GMP phosphodiesterases were determined at variuos stages of rat liver development. These results show (1) a period of increasing sensitivity of rat liver adenylate cyclase to glucagon at a time when sensitivity to NaF and Gpp(NH)p remains unchanged, and (2) increased responsiveness to glucagon plus Gpp(NH)p which is dependent upon the degree of glucagon sensitivity. It is concluded that the guanul nucleotide regulatory site is a functional part of adenylate cyclase very early in liver development and that the development of glucagon sensitivity is more probably limited by the developmet of glucagon receptors. Two forms of each phosphodiesterase (high and low K_m) were found throughout, except that low K_m cyclic GMP phosphodiesterase could not be demonstrated in the embryo. No significant change with age was found for the K_m or V of any of the enzyme forms. The ratio of soluble: particulate cyclic AMP phosphodiesterase decreased with age, whereas no change in the ration for cyclic GMP phosphodiesterase was observed. Specific activities of each enzyme from were highest in the perinatal period and decreased with age. The changes in phosphodiesterase specific activities paralled changes in guanylate and adenylate cyclase activities, which argues against a selective regulatory role for phosphodiesterase in modulating cyclic nucleotide influences during liver morphogenesis.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.