Enzyme Activity In Intact Cells Research Articles

Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe

Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation. To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with defined amounts of the probes' ceramide cleavage products and mounted them on lipid beads. Directly excited NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter

Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal β-hairpin motif that acts as both a ‘protectide’ to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The β-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (.CH(2)-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (> or =600 microm) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and .CH(2)-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.

A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia.

We investigated two novel point mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing a mild and a severe form of 3beta-HSD deficiency congenital adrenal hyperplasia. The first is a nonstop mutation in the normal stop codon 373 of the gene in exon IV [TGA (Stop) --> TGC (Cys) = Stop373C) identified from one allele of a female child with premature pubarche whose second allele had an E142K mutation. The Stop373C mutation predictably results in an open reading frame and a mutant-type (MT) II 3beta-HSD protein containing 467 amino acid residues, compared with the 372 amino acid residues of wild-type (WT) protein. The second is a homozygous missense mutation in codon 222 [CCA (Pro) --> ACT (Thr) = P222T] in the gene identified from a female neonate with salt-wasting disorder. The pcDNA vectors containing the constructs of WT II 3beta-HSD cDNA, WT cDNA with the open reading frame (WT cDNA(+)), MT Stop373C with the open reading frame (Stop373C(+)) and MT P222T cDNA were transfected in COS-I and 293T cells and expressed a similar amount of 3beta-HSD mRNA. The enzyme activity in intact cells using pregnenolone and dehydroepiandrosterone as substrate in the medium (1 micromol/liter) was identical between the WT cDNA and the WT cDNA(+), but was decreased to 27% of the WT enzymes at 6 h by MT Stop373C(+) enzyme, and was undetectable by P222T enzyme. In the homogenates of the cells, both MT Stop373C(+) and P222T enzyme activities and enzymes were undetectable despite clear detection of WT enzyme activities and WT enzymes. LH response to an LHRH analog stimulation in the pubertal female with the Stop373C/E142K genotypes and in a pubertal female with compound 273/318 frameshift genotypes were comparable to and higher than control females, respectively. In conclusion, a structurally lengthy MT II 3beta-HSD enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3beta-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3beta-HSD deficiency. The undetectable Stop373C and P222T enzymes on Western blottings, together with the respective in vivo and in vitro data, suggest that a relative instability of Stop373C enzyme and a profound instability of the P222T enzyme are likely the detrimental molecular mechanisms. The increased LH in the female with the frameshift genotype and the appropriate LH response in the female with the nonstop genotype correlated with predictably severe and mild ovarian type II 3beta-HSD deficiency, respectively.

Critical residues for the specificity of cofactors and substrates in human estrogenic 17beta-hydroxysteroid dehydrogenase 1: variants designed from the three-dimensional structure of the enzyme.

Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.

Influence of butyric and lactic acids on the beta-glucuronidase activity of Clostridium perfringens.

The beta-glucuronidase activity of intact cells of Clostridium perfringens was not influenced by the presence of either 0.09 or 0.19% lactic or butyric acids. In contrast, the enhanced enzyme activity of intact cells due to sodium deoxycholate was significantly decreased by the presence of these acids. These results suggest the possibility that the development of cancer due to the intake of a high fat diet may be inhibited by the presence of organic acids produced by intestinal bacteria.

Influence of various bile salts on beta-glucuronidase activity of intestinal bacteria.

While the decrease of the beta-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells.

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.

11β-Hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11β-hydroxysteroid dehydrogenases (11β-HSD) have been described. 11β-HSD-1 is NADP +-dependent and has a K m in the micromolar range and bidirectional activity. 11β-HSD-2 is NAD +-dependent, has a K m in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11β-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD +-dependent 11β-HSD-2. We found that the K m for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD +-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, K m = 40 nM, NADP +-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11β-HSD-1 nor the 11β-HSD-2 and have named it 11β-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5β-dihydroprogesterone, and 5α-dihydroprogesterone, but not bile acids.

Reversibility of retinoid effect on sialyltransferase activity, sialic acid content and invasive ability of human lung carcinoma cells : Ledinko N, Fazely F. Helene W. Toolan Institute for Medical Research, 110 Hospital Drive, Bennington, VT 05201. Anticancer Res 1989;9:1669-72

The effect of retinoid induced suppression of in vitro invasive ability of A549 human lung carcinoma cells on sialyltransferase activity and sialic acid content was investigated. Inhibition by retinol acetate of cell invasive potential was accompanied by a significant decrease in the enzyme activity of intact cells as well as total and cell surface neuraminidase-releasable sialic acid contents. Moreover, reversibility of the invasion-suppressed A549 cell phenotype resulted in a return of invasion potential, sialyltransferase activity and surface sialic acid content to invasive cell levels. These findings suggest that membrane-bound sialic acid plays a role in invasiveness of A549 cells.

Gamma-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization.

Escherichia coli cells showed maximum activity of gamma-glutamyltranspeptidase (EC 2.3.2.2) when they were grown at 20 degrees C, 14% of maximum activity at 37 degrees C, and none at 43 degrees C. The enzyme activity of intact cells grown at 20 degrees C was stably maintained after the temperature was changed to 45 degrees C. The activity increased during the exponential phase, and maximum activity was found at stationary phase. Its intracellular localization in the periplasmic space was confirmed.

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.

The presence of leucine aminopeptidase as an ecto-enzyme on the plasma membrane of neutrophils and its functions

5'-Nucleotidase, alkaline phosphodiesterase, Mg2+-ATPase and leucine aminopeptidase are generally reported to be located on the plasma membrane of various mammalian cell types. Among these enzymes, the presence of leucine aminopeptidase (LAP) in neutrophils has not been clear. Therefore, we started with examining the existence of LAP in neutrophils and its subcellular localization with reference to 5'-nucleotidase and alkaline phosphodiesterase. 5'-Nucleotidase was hardly detected in human. Alkaline phosphodiesterase was found in guinea-pig, human and rabbit, but no evidence for the ecto-enzyme was obtained in guinea pig. On the other hand, LAP was observed with all the neutrophils examined and proved to be located as an ecto-enzyme by applying the criteria DePierre and Karnovsky have used to define ecto-enzymes: comparison of the enzyme activity of intact cells with the total cell activity using sonicates, localization of the products of intact cell enzymatic activity and inhibition of intact cell enzyme by a “nonpenetrating” reagent without the inhibition of a soluble cytoplasmic enzyme. Next, the possible functions of LAP were examined. Tuftsin, a phagocytosis-stimulating tetrapeptide, is liberated from leukokinin, neutrophilic γ-globulin with phagocytosis-stimulating activity, by leukokininase on the plasma membrane of neutrophils and these stimulatory effects are not maintained and fall off in time to control levels. On the other hand, the biological activity of tuftsin is reported to be susceptible to commercial leucine aminopeptidase. Therefore, we examined whether LAP in neutrophils acts as a tuftsin-inactivating enzyme or not. Experimental data showed that a tuftsin-inactivating activity was localized as an ecto-enzyme on the plasma membrane and its activity was parallel with the change in the LAP activity which was competitively inhibited by tuftsin, suggesting the possible responsibility of LAP for the tuftsin-inactivating activity of neutrophils.Phagocytosis is characterized by internalization of large portion of surface membrane of neutrophils and so ecto-enzymes might be interiorized along with plasma membrane and inactivated during phagocytosis. Then, we studied changes in LAP activity during phagocytosis of phagocytic particles. Inactivation of LAP was observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of LAP activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor (s) which are probably activated by the attachement of an opsonized zymosan particle to a specific membrane receptor, probably the C 3 b receptor. Abovementioned results suggested that LAP may modulate the regulation of phagocytic function of neutrophils, which led us to investigate the distribution of the LAP activity among leukocytes other than neutrophils. So-called “professional” phagocytes such as neutrophils, monocytes and macrophages showed higher enzyme activity than lymphocytes and eosinophils in all species examined although the difference was observed in the LAP activity between species. A chemical modification of intact guinea-pig leukocytes with diazotized sulfanilic acid, a poorly penetrating reagent, suggested that LAP was located as an ecto-enzyme in all cell types.

Acyl-CoA:Cholesterol acyltransferase in chinese hamster ovary cells: Enzyme activity determined after reconstitution in phospholipid/cholesterol liposomes

Acyl-CoA:cholesterol acyltransferase from Chinese hamster ovary (CHO) cells was solubilized by deoxycholate, and then reconstituted in phosphatidylcholine/cholesterol liposomes. This reconstituted activity was totally dependent upon the cholesterol content of the mixture and showed saturation for cholesterol. Analysis of the reconstituted enzyme on linear Ficoll gradients shows that the enzyme has been incorporated into phospholipid/cholesterol liposomes. The CHO cell enzyme activity as measured by conventional assay (using cellular cholesterol as the substrate) was activated approximately 20-fold by low density lipoprotein. This activation process was independent of protein synthesis. When the above cell homogenates were assayed after optimal reconstitution, the activation produced by low density lipoprotein was essentially completely abolished. There was also no change in enzyme activity measured after reconstitution when cells were switched from sterol-containing medium to sterol-free medium, in contrast to a more than 7-fold drop in enzyme activity when assayed without reconstitution. These results suggest that the enzyme activity in intact cells is controlled by the content and composition of cellular lipids associated with the enzyme molecule. Since the intracellular messenger of low density lipoprotein is known to be cholesterol, it is likely that this enzyme activity in intact cells is primarily controlled by the cholesterol content in the vicinity of the enzyme molecule.

Stimulation of rat C6 glioma ecto-5'-nucleotidase by chronic ethanol treatment

Treatment of rat C6 glioma cells in culture with 100 mM ethanol led to increased ecto-5'-nucleotidase activity. Studies were performed to obtain information on the mechanism(s) of action for this effect. Growth of C6 glioma cells in 100 mM ethanol for 6 days increased ecto-5'-nucleotidase activity but had no effect on the pH optima or substrate specificity of the ecto-enzyme. A comparison of enzyme activity of intact cells with that of disrupted cells revealed a selective stimulation of the ecto-enzyme, i.e. the enzyme on the outer plasma membrane. Kinetic studies of cells continuously treated with 100 mM ethanol for 6 days showed an apparent increase in affinity of the ecto-enzyme for 5'-AMP, with no appreciable change in the maximum velocity ( V max). The concentration-dependent inhibition of ecto-5'-nucleotidase activity by Concanavalin A (Con A) was antagonized by ethanol treatment. Double reciprocal plot analysis showed that ethanol acted in an apparently noncompetitive manner with respect to Con A inhibition. It appears that the availability of the catalytic site of the ecto-5'-nucleotidase to exogenous substrates was increased. The data are consistent with rearrangement of the plasma membrane resulting from continuous exposure to ethanol.

Altered ribonucleotide reductase activity in drug-resistant mammalian cells detected by an assay procedure for intact cells.

Hydroxyurea, an antitumor drug which specifically inhibits ribonucleotide reductase, has been used as a selective agent to obtain single-step and double-step drug-resistant cell lines altered in reductase activity. Using an assay procedure developed for analyzing enzyme activity in intact cells a variety of novel changes in the properties of the enzyme activity was detected in the drug-resistant lines. Differences in cellular levels of enzyme activity, changes involving pyrimidine and purine substrate Km values, and altered enzyme sensitivity to hydroxyurea were found using the in vivo assay procedure. These changes could account for the drug-resistant phenotypes. The assay technique which is described in this report is easy to perform, can accurately determine enzyme activity in as few as 5 X 10(6) cells grown conveniently on the surface of a single tissue culture plate, and is a valuable quick screening method for identifying cells altered in different aspects of ribonucleotide reductase activity.

Two methods for determining the activity of δ-aminolevulinate synthetase within intact liver cells in culture

Two methods are described by which δ-aminolevulinate synthetase activity is assayed within cultures of intact chick embryo liver cells. The methods depend on blocking further metabolism of δ-aminolevulinate with levulinate (25 m m). Under these conditions, the δ-aminolevulinate accumulates, much of it leaking out into the medium. The δ-aminolevulinate is measured colorimetrically in a 10-cm light-path cuvette. As little as 0.5 mg of cell protein/assay is required, and the lower limit of the spectrophotometric assay is 600 pmol/2 ml of culture medium. With the first method, the accumulation of δ-aminolevulinate can be followed during the induction, by chemicals, of δ-aminolevulinate synthetase within the intact cell. The second method serves as a quantitative assay for the amount of enzyme present at any one time point. Method 1 is sufficiently sensitive to measure the low activity of the uninduced enzyme as the accumulation of δ-aminolevulinate over a period of 16 hr. With cells in which δ-aminolevulinate synthetase has been chemically induced, method 1 yields a rate of δ-aminolevulinate production by these intact cells, 50–60% of the rate of δ-aminolevulinate produced by homogenates of the same cells incubated in an optimal assay medium. Glycine added to the culture medium at 5 m m increased by three-fold δ-aminolevulinate production by the induced intact cells. With method 2, the enzyme activity of intact cells which have been induced for a defined time, can be measured merely by replacing the culture medium with an “assay medium” containing levulinate (30 m m), glycine (50 m m), citrate (5 m m) and buffer and then incubating the cells for 5 hr. During this time, δ-aminolevulinate accumulates at a linear rate. The rate is equivalent to that of the homogenates of the same cells. The advantage of method 2 compared with the assay of homogenized cells is that the enzyme can be assayed in small amounts of cells without harvesting. One 3.5-cm dish containing 0.7 mg of cell protein is sufficient for one determination.

Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since alpha-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium.

The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Induction by vitamin K

An enzyme present in cell-free extracts of B. melaninogenicus grown with vitamin K is described which catalyzes the synthesis of 3-ketodihydrosphingosine from palmitoyl CoA and l-serine. Activity of the 3-ketodihydrosphingosine synthetase was measured as a function of time, palmitoyl CoA concentration, and serine concentration. The Bacteroides synthetase differs from corresponding enzymes from brain microsomes and from yeast in that it is present in the 100,000 g supernatant of sonicated cells and is not associated with any particulate fraction. Extracts prepared from cells depleted of vitamin K showed only slight 3-ketodihydrosphingosine synthetase activity. Neither vitamin K 1, menadione, nor pyridoxal phosphate were effective in enhancing the activity in cell-free extracts of vitamin K-depleted B. melaninogenicus. However, induction of the enzyme activity in intact cells was demonstrated by the addition of vitamin K to a vitamin K-depleted culture. Synthetase activity was found to be increased 15 min following the addition of the vitamin, reached a maximum at 75 min, and thereafter remained constant. Both puromycin and rifampcin inhibit induction of the enzyme by vitamin K 1 suggesting that vitamin K induces de novo synthesis of the synthetase.