In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae.

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model. The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.     

Ornithine-σ-Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture

To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.     

Assay of glyoxalase I in blood

Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was assayed using alcoholic, acidic 2,4-dinitrophenylhydrazine to follow the disappearance of methylglyoxal over time, with the absorbance of formed methylglyoxal bis-hydrazone measured at 432 nm. Erythrocyte glyoxalase I activities were found to be 64, 41, and 18 mumole of S-lactoyl glutathione formed min-1 X ml-1 of red blood cells in rat, human, and rabbit blood and 174 mumole X min-1 X mg-1 of protein for yeast. The Km values found in millimolar hemimercaptal were about 0.5. Glyoxalase I activity can be determined in crude tissue preparations without interference from biological materials.     

Membrane-bound succinate dehydrogenase of Bacillus pumilus strain 5: effects of modulators of monoelectron transfer

The membrane-bound succinate dehydrogenase (SDH; EC 1.3.99.1) of Bacillus pumilus strain 5 was investigated as succinate:ferricyanide oxidoreductase activity at 27 degrees C. A Km of 8.3 x 10(-3) M was obtained, and the Vmax was 1.8 x 10(-6) mole succinate dehydrogenated min-1 mg-1 membrane protein, at a substrate (succinate) concentration below 40 x 10(-3) M. Above this succinate concentration the Km was 102 x 10(-3) M and the Vmax was 3.7 x 10(-6) mole succinate min-1 mg-1 membrane protein. Para-benzoquinone or 2,4-dinitrophenylhydrazine, in micromolar amounts inhibited the enzyme by serving as an electron sink. Hydroxyl radical (OH.) scavengers, mannitol and benzoate, activated the enzyme, while superoxide dismutase (SOD) had no effect on the enzyme. Thus, the mechanism of electron transfer from succinate to Fe(CN)3-(6) through SDH does not involve superoxide (O2-) as a rate-limiting intermediate.     

Purification and characterization of an alpha-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280.

A novel alpha-glucosidase with an apparent subunit mass of 59 +/- 0. 5 kDa was purified from protein extracts of Rhizobium sp. strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 +/- 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35 degrees C. The activity increased in the presence of NH4+ and K+ ions but was inhibited by Cu2+, Ag+, Hg+, and Fe2+ ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl alpha-glucoside was the fluorogenic substrate. The enzyme was more active with alpha-glucosides substituted with aromatic aglycones than with oligosaccharides. This alpha-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl alpha-D-glucopyranoside (Km, 0.141 microM; Vmax, 6.79 micromol min-1 mg-1) and with p-nitrophenyl alpha-D-glucopyranoside (Km, 0.037 microM; Vmax, 2.92 micromol min-1 mg-1). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Red blood cell lactate transport in sickle disease and sickle cell trait.

This study determined and compared rates and mechanisms of lactate transport in red blood cells (RBCs) of persons with 1) sickle cell disease (HbSS), 2) sickle cell trait (HbAS), and 3) a control group (HbAA). Blood samples were drawn from 30 African-American volunteers (10 HbSS, 10 HbAS, 10 HbAA). Lactate influx into RBCs was measured by using [14C]lactate at six (2, 5, 10, 15, 25, and 40 mM) unlabeled lactate concentrations. The monocarboxylate transporter pathway was blocked by p-chloromercuriphenylsulfonic acid to determine its percent contribution to total lactate influx. Generally, total lactate influx into RBCs from the HbSS group was significantly greater than influx into RBCs from HbAS or HbAA, with no difference between HbAS and HbAA. Faster influx into HbSS RBCs was attributed to increased monocarboxylate transporter activity [increased apparent Vmax (V'max)]. V'max (4.7 +/- 0.6 micromol x ml(-1) x min(-1)) for HbSS RBCs was significantly greater than V'max of HbAS RBCs (2.9 +/- 1.5 micromol x ml(-1) x min(-1)) and HbAA RBCs (2.0 +/- 0.5 micromol x ml(-1) x min(-1)). Km (42.8 +/- 8 mM) for HbSS RBCs was significantly greater than Km (27 +/- 12 mM) for HbAA RBCs. We suspect that elevated erythropoietin levels in response to chronic anemia and/or pharmacological treatment (erythropoietin injections, hydroxyurea ingestion) is the underlying mechanism for increased lactate transport capacity in HbSS RBCs.     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.     

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.     

Sexual response in Saccharomyces cerevisiae: alteration of enzyme activity in the glyoxalase system by mating factor

Glyoxalase I activity in alpha-type budding yeast of the Saccharomyces cerevisiae strain was increased by exposure of alpha-type cells to supernatant of a culture of a-type yeast cells, although glyoxalase II activity was decreased by the same treatment. The alteration of enzyme activity in the glyoxalase system occurred during the 30-60 min period after exposure of alpha-type cells to a-type culture supernatant. No change of glyoxalase I and II activities was found in the case of the alpha-type strain, S. cerevisiae VQ3 (alpha ste3-1), which is deficient in a-factor receptors.     

Human microsomal prostaglandin E synthase-1: purification,functional characterization,and projection structure determination

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol min-1 mg-1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.     

Differential behaviour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi

1. Glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) of two morphological forms of Trypanosoma cruzi, epimastigotes and metacyclics, are reported. 2. The kinetic behaviour and some of the kinetic parameters of the enzyme in both forms were studied. The enzymes showed a simple Michaelis-Menten kinetic. 3. The activity in epimastigote forms was alway higher than the metacyclic ones. At subsaturating concentrations of substrate was almost 10-fold higher, whereas at saturating concentrations was about 2-fold higher. 4. In epimastigote forms the specific activity and Km values, at pH 7.5 and 37 degrees C, was found to be 142 mUnits x mg-1 of protein and 0.23 mM, respectively. 5. In the same conditions, the specific activity and Km values in metacyclic forms was 75 mUnits x mg-1 of protein and 1.06 mM, respectively. 6. A possible role in the carbohydrate metabolism of glucose 6-phosphate dehydrogenase in both forms of Trypanosoma cruzi is discussed.     

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.

To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase.     

Purification and characterization of S-adenosylhomocysteine hydrolase from mouse mastocytoma P-815 cells. Evidence for cell cycle-specific fluctuation of the enzyme activity

S-Adenosylhomocysteine hydrolase [EC 3.3.1.1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190,000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45,000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The Km value for adenosine was 0.29 microM and the Vmax value 4.5 mumol S-adenosylhomocysteine X min-1 X mg-1 in the synthetic reaction, while the Km value for S-adenosylhomocysteine was 0.77 microM and the Vmax 0.48 mumol adenosine X min-1 X mg-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (KD = 2.61 X 10(-7) M) and one for cAMP (KD = 1.6 X 10(-7) M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G1/S phase to the G2 phase.     

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.     

Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product.

Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5- (and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 x 10(-8) m s-1. Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40 degrees C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s-1 was calculated. Hydrolysis of cFDA (at 40 degrees C) in cell extracts was described by Michaelis-Menten kinetics with an apparent Vmax and Km of 12.3 nmol.min-1.mg of protein-1 and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed.