Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Deoxyguanosine kinase from human placenta

Deoxyguanosine kinase (ATP:deoxyguanosine 5'-phosphotransferase) has been purified up to a specific activity of 10.3 nmol/min per mg protein from human placenta. The enzyme appears to have a molecular weight of 58 000 from the results of Sephadex G-75 gel filtration. The enzyme catalyzed phosphorylation of deoxyguanosine and deoxyadenosine, but deoxycytidine was not phosphorylated. An apparent Km value for deoxyguanosine was 2.5 micro M. When ATP was used as a phosphate donor, the pH optimum was at pH 6.0, but the optimum was shifted to pH 6.8 by the addition of dTTP. At physiological pH, the activity was stimulated 3-4-fold by dTTP. dTTP was also an effective phosphate donor, but using dTTP as a phosphate donor, a broad pH optimum of 7.0 was observed. Two Km values of 0.13 and 2.2 mM were obtained for both MgATP2- and MgdTTP2-. The activity was strongly inhibited by dGTP and dGDP; 50% inhibition by 1.0 micro M dGTP and 2.1 micro M dGDP, respectively. The enzyme required the presence o Mg2+ or Mn2+.     

Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).     

Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide to a study of the inhibition by leupeptin.

1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4--6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3--6.0, with the mean value 7 x 10(-9) M.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.     

Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme

The presence of two forms (high and low molecular weight ones) of purine nucleoside phosphorylase II (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was demonstrated. The high molecular weight form of the enzyme was purified, and the properties of both forms were compared. The enzyme forms were shown to differ in their quaternary structure (trimeric and hexameric), molecular weight of the native enzyme and its subunits (85,000 and 28,000 for the trimer, 150,000 and 25,000 for the hexamer, respectively) as well as substrate specificity (the trimer is specific for all major purine nucleosides, while the hexamer does not cleave adenine nucleosides). Adenosine is a competitive inhibitor of the hexameric form with respect to deoxyguanosine (Ki = 1.16 X 10(-3) M); the Km value for deoxyguanosine is 9.85 X 10(-5) M. The isoelectric point for the both forms of the enzyme in the presence of 9 M urea is about 5.5. Both forms have a pH optimum of phosphorolytic activity between 6.5 and 7.0.     

Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine.

Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

The effect of several 2-alkyl- and 4'-O-methylanalogs of pyridoxol on mouse liver pyridoxal kinase activity]

A simple method of isolation of partially purified puridoxal kinase preparation from mouse liver, having specific activity of 600-700 E/mg protein and a 30% yield is described. It is demonstrated that of all number of 2-alkyl- and 4'-O-methyl pyridoxol analogs synthesized, 4'-O-methyl-pyridoxol (Ki=0.2-10(-5) M, Km(pyridoxal)=4-10(-5) M) is the most active competitive inhibitor of pyridoxal kinase. 3-Deoxy-4'-O-methylpyridoxol is a non-competitive inhibitor of pyridoxal kinase, the latter having an affinity for the enzyme 16 times lower than that of 4'-O-methylpyridoxol. 2-Alkyl analogs of pyridoxol exhibit properties of competitive inhibitors; the affinity of 2'-ethylpyridoxol for the enzyme is 5 times lower than that of 2'-methylpyridoxol; corresponding 2-alkyl derivatives of dimethyl ethers of 3-hydroxycinchomeronic acids have no pronounced affinity for the enzyme. The study of the toxic effects of pyridoxol analogs on the central nervous system has revealed inverse dependence between the neurotoxic dose of the compound and its efficiency as an inhibitor of pyridoxal kinase (Km/Ki value).     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.     

Adenosine deaminase: physical and chemical properties of partially purified mitochondrial and cytosol enzyme from rat liver.

Adenosine deaminase (ADA) was partially purified 486- and 994-fold from rat liver mitochondria and cytosol, respectively. Relative molecular mass of the enzymes from both fractions was 34,000. Km for adenosine and 2'-deoxy-adenosine were 3.08 x 10(-5) M and 3.03 x 10(-5) M for mitochondrial ADA and 3.12 x 10(-5) M and 2.87 x 10(-5) M for cytosolic ADA. The enzyme from both subcellular fractions had the maximum activity at pH 7.5-8.0, and pI 5.2 and 4.2 for mitochondrial and cytosolic enzyme, respectively. The enzyme was inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine and 2'-deoxycoformycin with Ki 4.4 x 10(-7) M and 3.2 x 10(-7) M for mitochondrial ADA and 4.9 x 10(-7) M 2.8 x 10(-7) M for cytosolic ADA. Among the natural nucleoside and deoxynucleotide derivatives tested, deoxy-GTP and UTP inhibited only cytosolic adenosine deaminase by 60% and 40%, respectively.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.     

Characteristics of delta 1-pyrroline-5-carboxylate reductase from Drosophila melanogaster.

1. Biochemical properties of delta 1-pyrroline-5-carboxylate reductase from d. melanogaster have been investigated. 2. The enzyme is stable below 4 degrees C. 3. the pH optimum of the enzyme is 5.7. It is rapidly inactivated below pH 5.4. 4. The Km values for NADPH and delta 1-pyrroline-5-carboxylate are 1.6 x 10-5 and 2.5 x 10-6 M, respectively. 5. the estimated molecular weight of the enzyme is 225,000. 6. the enzyme is weakly inhibited by L-proline (Ki = 0.12 M).     

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography.

An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10(-5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.     

Inhibition of mouse spleen inorganic pyrophosphatase by methylene diphosphonic acid]

Some properties of mouse spleen cytosol inorgainc pyrophosphatase (PPi-ase) (E. C. 3.6.1.1) as well as the effect of methylene diphosphonic acid (PCP) on the PPi-ase activity were studied. Specific staining for the enzyme PAAG disc-electrophoresis was developed; it was shown that the PPi-ase formed only one band in 7.5% PAAG. The enzyme pH optimum being 8.0, the optimal [Mg++]/[PPi] ratio was about 2; Km =7.7x10(-4) M, Vmax=0.77 mkM. min-1. mg protein-1. PCP was shown to competitively inhibit the pyrophosphatase reaction, Ki=2.5x10(-4) M +/- 0.2x10(-4) M.