Biochemical properties of trypanosomatid lactate dehydrogenases

Lactate dehydrogenase (LDH, E.C.1.1.1.27) was found in supernatant (cytoplasmic enzyme) fractions of the trypanosomatid flagellates Trypanosoma conorhini and Crithidia fasciculata if 10 mm cysteine was present in the homogenizing medium. The T. conorhini LDH activity with pyruvate as substrate was increased 35% if 5 mm cysteine was also included in reaction mixtures. K(m) values for the T. conorhini enzyme were 3.3 x 10(-4)m with pyruvate, and 1.6 x 10(-4)m with alpha-ketobutyrate. Cysteine inhibited alpha-ketobutyrate reduction. Comparison of trypanosomatid and human serum LDH enzymes with respect to K(m), substrate activity and inhibition, pH optima, and K(i) values for oxalate and oxamate indicated that the trypanosomatid isoenzymes differed significantly from serum LDH. C. fasciculata LDH was extremely labile, since 59% of the activity was lost 90 min after isolation. The role of LDH enzymes in trypanosomatid metabolism is discussed, and the results are related to other trypanosomatid LDH enzymes. The comparison of homologous enzymes in host and parasite is discussed with regard to metabolic function and a possible model system for chemotherapy.     

Partial purification and characterization of dihydrodipicolinic acid synthetase from sporulating Bacillus megaterium

Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.     

Purification and properties of the adenosine diphosphate-glucose and uridine diphosphate-glucose pyrophosphorylases of Mycobacterium smegmatis: inhibition and activation of the adenosine diphosphate-glucose pyrophosphorylase

Crude extracts of Mycobacterium smegmatis catalyzed the synthesis of adenosine diphosphate-glucose (ADP-Glc), cytidine diphosphate-glucose, guanosine diphosphate-glucose (GDP-Glc), thymidine diphosphate-glucose (TDP-Glc), and uridine diphosphate-glucose (UDP-Glc). In these crude enzyme fractions, high concentrations of trehalose-P inhibited the ADP-Glc and GDP-Glc pyrophosphorylases but did not effect the UDP-Glc or TDP-Glc pyrophosphorylases. Both the ADP-Glc pyrophosphorylase and the UDP-Glc pyrophosphorylase were partially purified (about 140-fold and 60-fold, respectively), and their properties were compared. For the ADP-Glc pyrophosphorylase, the K(m) for adenosine triphosphate was 6 x 10(-4)m, whereas that for glucose-1-P was 8 x 10(-4)m. The optimal concentration of Mg(2+) was 1 x 10(-3)m, and the pH optimum was 8.5. For the UDP-Glc pyrophosphorylase, the K(m) for uridine triphosphate was 1 x 10(-3)m and for glucose-1-P was 2 x 10(-3)m. The optimal Mg(2+) concentration was 1 x 10(-3)m, and the pH optimum was about 8.0. The purified ADP-Glc pyrophosphorylase was inhibited by fructose-6-P, fructose-1, 6-diphosphate, glucose-6-P, and phosphoenolpyruvate. On the other hand, trehalose, trehalose diphosphate, sodium pyruvate, and ribose-5-P did not effect the ADP-Glc pyrophosphorylase. None of these compounds, including trehalose-P, had any effect on the UDP-Glc pyrophosphorylase.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a K(m) of 4.26x10(-5)m for RS-mevalonate and 1.54x10(-3)m for ATP. The preparation from green leaves had a K(m) of 4.55x10(-5)m for RS-mevalonate and 1.75x10(-3)m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.     

Phanerochaete chrysosporium beta-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate beta-glucosidases and an extracellular beta-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K(m) for p-nitrophenyl-beta-glucoside is 1.6 x 10 M; the K(i) for glucose, a competitive inhibitor, is 5.0 x 10 M. The K(m) for cellobiose is 5.3 x 10 M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K(m) for p-nitrophenyl-beta-glucoside is 1.1 x 10 M. The molecular weight of this enzyme is approximately 410,000. Both enzymes have an optimal temperature of 45 degrees C and an E(act) of 9.15 kcal (ca. 3.83 x 10 J). The pH optima, however, were approximately 7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

Kinetic properties of cerebral pyruvate kinase

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.     

Characterization and properties of the pyruvate phosphorylation system of Acetobacter xylinum

The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning

Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.     

Properties and purification of N-acetylmuramyl-L-alanine amidase from Staphylococcus aureus H

The principal autolytic enzyme activity of the cell sap of Staphylococcus aureus H has been purified 400-fold. It is an N-acetylmuramyl-l-alanine amidase. This enzyme has a molecular weight of 8 to 10 x 10(5), a pH optimum of 7.3, an ionic strength optimum of 0.16 m and a K(m) of 10(-3)m murein repeating units.     

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.     

Lytic enzyme from lysates of Streptomyces venezuelae infected with actinophage MSP2

A lytic enzyme was purified 600-fold with 12% recovery from lysates of Streptomyces venezuelae S13 infected with actinophage MSP2. The purified enzyme preparation was homogeneous as shown by polyacrylamide electrophoresis. The enzyme was active over a pH range 6.0 to 9.0 with a maximum at pH 7.5. The pH profile for stability was sharp, with an optimum at pH 7.5. Maximal activity occurred between 30 and 35 C. The enzyme was stable at 20 C or less. A 30-min exposure to 25, 30, 35, 40, 45, and 50 C produced an inactivation of 3, 40, 77, 82, 93, and 100%, respectively. Lytic activity was stimulated fivefold by either 5 x 10(-3)m Mg(2+) or Mn(2+) and three- and twofold by Ca(2+) and Ba(2+), respectively. Addition of Na(+), K(+), NH(4) (+), or Li(+) to the tris(hydroxymethyl)aminomethane-hydrochloride buffer did not alter the rate of lysis. Enzyme activity was inhibited 74 and 27% by 10(-4) and 10(-5)m ethylenediaminetetraacetic acid (EDTA), respectively. The inhibition by EDTA was reversed partially by addition of Mg(2+). Lytic activity was abolished by either 5 x 10(-4)m HgCl(2) or p-hydroxymercuribenzoate, whereas 5 x 10(-4)m CuSO(4) inhibited 72%. Cell wall solubilization paralleled the release of N-terminal amino groups and reached a level of 0.23 mumole per mg of cell walls. No release of reducing power was detected in treated or untreated cell wall suspensions. Tests for proteolytic activity were negative.     

Fluorimetric determination of d-amino acid oxidase

1. A sensitive fluorimetric procedure for the assay of d-amino acid oxidase has been developed. 2. The method depends on the formation of a fluorescent derivative, 2-hydroxy-3-methylquinoxaline, on condensation of pyruvate with o-phenylenediamine in acid medium. 3. 2-Hydroxy-3-methylquinoxaline fluoresces strongly in 50% (v/v) sulphuric acid after excitation at 375mmu. A single emission peak is observed at 480mmu. 4. Formation of the quinoxaline is dependent on time, temperature, acidity and relative concentration of reactants. 5. A particulate preparation from mouse kidney required FAD for optimum activity at pH8.5; K(m) was 3.8x10(-3)m; K(FAD) was 1.4x10(-7)m and the reaction was strongly inhibited by p-chloromercuribenzoate and phenylmercuric acetate. 6. Subcellular fractionation on a sucrose density gradient confirmed that the d-amino acid oxidase was localized on small granules.     

Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).     

The purification and properties of histidinol dehydrogenase from Neurospora crassa

1. A procedure is described for the purification of l-histidinol dehydrogenase (l-histidinol-NAD oxidoreductase, EC 1.1.1.23) from Neurospora crassa. 2. The enzyme, as purified, has a sedimentation coefficient, S(20), of 7.1s and a molecular weight of 81 000. Considerable variation is possible in the state of polymerization of the enzyme, giving rise to observed molecular weights from 40 000 to 240 000. 3. Several kinetic parameters of the enzyme have been determined. The enzyme is maximally active at pH9.8; the K(m) (NAD) is 13.0x10(-5)m and K(m) (histidinol) is 8.2x10(-6)m. The enzyme is highly specific, does not oxidize a range of amino alcohols and other aliphatic alcohols nor reduce NADP and has no demonstrable affinity for histidine. The turnover number is 49 moles of NAD reduced/min./mole of enzyme (mol.wt. 40 000).     

S-Adenosylmethionine decarboxylase from human prostate. Activation by putrescine

1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent K(m) for the substrate is 4x10(-5)m. The calculated S-adenosylmethionine content of human prostate (0.18mumol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0 degrees C and is rapidly inactivated at 40 degrees C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-(14)C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6'-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of ;essential' thiol groups.