Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Metabolic adaptation of the renal carbohydrate metabolism. III

Summary The adaptive response of renal metabolism of glucose was studied in isolated rat proximal and distal renal tubules after a high protein-low carbohydrate diet administration. This nutritional situation significantly stimulated the gluconeogenic activity in the renal proximal tubules (about 1.5 fold at 48 hours) due, in part, to a marked increase in the fructose 1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. In this tubular fragment, FBPase activity increased only at subsaturating fructose 1,6-bisphosphate concentration (30% at 48 hours) which involved a significant decrease in the Km (31%) for its substrate without changes in the Vmax. This enzymatic behaviour is probably related to modifications in the activity of the enzyme already present in the renal cells. Proximal PEPCK activity progressively increased at all substrate concentrations (almost 2 fold at 48h of high protein diet) which brought about changes in Vmax without changes in Km. These changes are in agreement with variations in the cellular concentration of the enzyme. Neither gluconeogenesis nor the gluconeogenic enzymes changed in the distal fractions of the renal tubules. On the other hand, a high protein diet did not apparently modify the glycolytic ability in any fragment of the nephron, although a significant increase in the phosphofructokinase (PFK) and pyruvate kinase (PK) activities was found in the distal renal tubules. This short term regulation involved a significant decrease from 24 hours in the Km value of distal PFK (almost 40%) without changes in Vmax. The kinetic behaviour of distal PK was mixed. In the first 24h after high protein diet a significant decrease in the Km for phosphoenolpyruvate was found (30%) without variation in the Vmax, however during the second 24 hours the activity of this glycolytic enzyme increased significantly (almost 1.3 fold) without modifications in its Km value. On the contrary, this nutritional state did not modify the kinetic behaviour of any glycolytic enzyme in the proximal regions of the renal tubules.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.     

Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells

Km values for H2O2 and Vmax values for three types of myeloperoxidase (MPO) from human leukocytes (MPO-I, -II, and -III) and four types from human myeloid leukemia HL-60 cells (MPO-IA, -IB, -II, and -III) were determined. Km values of human leukocyte MPOs decreased with increasing pH from 4.4 to 6.2 and increased with increasing NaCl concentration from 0.025 to 0.14 M. There was no significant difference among Km values of leukocyte MPO-I, -II, and -III. NaBr also showed a tendency similar to that of NaCl with regard to the effects of pH and halide concentration on Km values. However, Km values in the presence of NaBr were lower than those in the presence of NaCl. Effects of pH and NaCl concentration on Vmax values of MPO-I, -II, and -III were also examined. Vmax values of MPO-I, -II, and -III were higher at pH 4.9 and 5.4 and increased with increasing NaCl concentration. In addition, no difference was observed between Km values of leukocyte and those of HL-60 cells. MPO-IB, the half-molecular-weight enzyme of HL-60 cells, also had the same Km values as the others. Furthermore, inhibition of the activities of seven MPOs of leukocytes and HL-60 cells by H2O2 was similarly observed at concentrations above 1 mM at pH 5.4 but not at pH 4.4. These results indicate that there is no difference in the affinity to H2O2 among leukocyte MPO-I, -II, and -III and HL-60 cell MPO-IA, -IB, -II, and -III.     

Stimulation of tyrosine protein kinase activity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) stimulated the activity of a partially purified tyrosine protein kinase from rat lung. The stimulation was concentration dependent with a maximum stimulation (about 2 fold) observed at 10 per cent (V/V) DMSO. On the other hand, acetone (10 percent, V/V), did not exert any stimulatory effect on the enzyme activity. The stimulation was associated with a decrease in the Km for the substrate and an increase in the Vmax. In contrast, the Km for ATP was not affected by DMSO. Under identical assay conditions, DMSO did not significantly alter the activities of phosphorylase kinase, catalytic subunit of cAMP-dependent protein kinase and Ca2+-phospholipid-dependent protein kinase. It may be speculated that stimulation of tyrosine protein kinase may be one of the mechanisms by which DMSO exerts its biological effects.     

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.     

Properties of the 12-O-Tetradecanoylphorbol-13-Acetate-Stimulated S6 Kinase from Rat Astroglial Cells

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

Sodium ion-stimulated alpha-[1-14C]aminoisobutyric acid uptake in alkalophilic Bacillus species.

Alkalophilic Bacillus no. 8-1 grows well in alkaline media containing 2.5 to 5% NaCl. The uptake of alpha-aminoisobutyric acid (AIB) into the cells is stimulated by the addition of NaCl (Na+) up to a concentration of 0.2 M, but other monovalent cations such as K+, Li+, or NH4+ cannot substitute for Na+. The kinetic studies reveal that, when the Na+ concentration increases from 0.02 to 0.2 M in alkaline medium, the Km for transport decreases, whereas Vmax remains almost constant. Competition studies indicate that glycine, L-alanine, L-serine, and AIB share common carriers for the transport of the compounds into cells. Other alkalophilic bacteria are also found to require Na+ for the uptake of AIB into the cells.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Purification and characterization of cell-associated glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.     

Sodium and ATP affinities of the cardiac (Na,K)-ATPase in relation to nitric oxide synthesis in spontaneously hypertensive rats

The (Na,K)-ATPase is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembraneous efflux of Na(+) from cardiac cells in spontaneously hypertensive rats (SHR) with increased synthesis of nitric oxide (NO). In the investigated group of SHR the systolic blood pressure was increased by 64% and the synthesis of NO was increased by 60% in the heart. When activating the cardiac (Na,K)-ATPase with substrate, its activity was higher in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed an increase of the V(max) (by 37%) probably due to increased affinity of the ATP-binding site as indicated by the lowered K(m) value (by 38%) in SHR. During activation with Na(+), we observed no change in the enzyme activity below 10 mmol/l of NaCl whereas in the presence of higher concentrations of NaCl the (Na,K)-ATPase was stimulated. The value of V(max) increased (by 64%), however the K(Na) increased (by 106%), indicating an adaptation of the Na(+)-binding site of the enzyme to increased [Na(+)](i). Thus the (Na,K)-ATPase in our SHR group is able to extrude the excessive Na(+) from myocardial cells more effectively also at higher [Na(+)](i), while the enzyme from controls is unable to increase its activity further. This improvement of the (Na,K)-ATPase function is supported also by increased affinity of its ATP-binding site probably due to enhanced NO-synthesis.     

Angiotensin I-converting enzyme from guinea pig lung and serum. A comparison of some kinetic and inhibition properties.

The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar.     

Effect of salts on D-glycerate dehydrogenase kinetic behavior.

Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Characterization of UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B.

UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1, 6-N-acetylglucosaminyltransferase V (GlcNAcT-V) has been purified from cell extracts of the human hepatoma cell line, Hep3B, with 8.7% recovery. The purified enzymes had molecular masses of about 67 and 65 kDa on denaturated and natural conditions, respectively. The values of pI was 5.9. The GlcNAcT-V, when resolved by SDS-PAGE, was positive for Schiff staining, suggesting that the enzyme is glycoprotein. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzyme displayed a temperature optimum of around 50 degrees C and optimum an pH of 6.5. The enzyme was stable in response to incubation from pH 4.5 to pH 10.5 at 4 degrees C for 24 h. The presence of UDP-GlcNAc and GlcN,GlcN-bi-PA protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ion; however, it was inhibited by Fe3+. The enzyme activity was inhibited by another series of NDP-sugars including ADP-, CDP-, GDP-, and TDP-GlcNAc. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward biantennary (GlcN,GlcN-bi-PA) sugars. The enzymes had apparent Km values of 1.28 and 5.8 mM for GlcN,GlcN-bi-PA and UDP-GlcNAc, respectively. In order to isolate the GlcNAcT-V gene, PCR primers of GNN-1 and GNN-8 were designed and the amplified PCR product carrying the gene was cloned and sequenced. Nucleotide sequence analysis showed a 2220-bp open reading frame encoding a 740-amino-acid protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. The three amino acid changes were as follows: 375V --> L, 555T --> R, and 592A --> G. These studies represent the detailed characterization of a purified GlcNAcT-V from human hepatoma cell Hep3B.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.