Evidence for a histidine and a cysteine residue in the substrate-binding site of yeast alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5'-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5'-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.     

Purification and properties of a kininogenin from the venom of Vipera ammodytes ammodytes.

A kininogenin (EC 3.4.21.8) was purified from the venom of Vipera ammodytes ammodytes (European sand viper) by a combination of gel filtration and ion-exchange chromatography. The enzyme is approximately six times more active than bovine trypsin in its ability to release vasoactive peptides from a plasma precursor. The kininogenin is a glycoprotein containing 18-20% by weight of carbohydrate. It showed a mol. wt. of 40500 on gel filtration. Gel electrophoresis of the reduced sample in the presence of sodium dodecyl sulphate and 2-mercaptoethanol revealed the presence of two major components of mol.wt. 34300 and 31300. The heterogeneity, which was also observed on disc electrophoresis, was removed by incubation with neuraminidase. After incubation with neuraminidase the kininogenin retained full enzymic activity and possessed an isoelectric point of pH7.2. The carbohydrate content has been decreased to 10% by weight, and the single component seen on electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol corresponded to a mol.wt. of 29500.     

Nuclear phosphoprotein phosphatase from calf liver.

Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.     

Effect of O-sulphate groups in lactose and N-acetylneuraminyl-lactose on their enzymic hydrolysis.

1. Lactose 6''-O-sulphate, N-acetylneuraminyl-(alpha 2 leads to 3)-D-lactose 6''-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 3)-D-lactose 6''0-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 6)-D-lactose and N-acetylneuraminyl-(alpha 2 leads to 3)- and -(alpha 2 leads to 6))-lactose 6''-O-sulphate were prepared by chemical sulphation of lactose, N-acetylneuraminyl-lactose and tis isomers by using pyridine-SO3 reagent. 2. Significant kinetic differences were observed in the enzymic hydrolysis of the sulphated derivatives compared with unsubstituted substrates. 3. In the case of reactions catalysed by rat liver lysosomal and Clostridium perfringens neuraminidases (EC 3.2.1.18), the presence of an O-sulphate group in the N-acetylneuraminyl moiety affected the reaction by decreasing the Km and the Vmax, its presence in the galactosyl moiety affected the reaction by decreasing the Km and increasing the Vmax. and its presence in both N-acetylneuraminyl and galactosyl moieties decreased the Km and the Vmax. of the reaction. 4. Mixed-substrate reaction kinetic data indicated competition between the sulphated and unsubstituted substrates for the same active sites on the neuraminidase molecule. 5. Lactose 6''-O-sulphate neither behaved as a substrate nor acted as an inhibitor with respect to unsubstituted lactose and p-nitrophenyl beta-D-galactopyranoside when tested with lactase of suckling rat intestine and Escherichia coli beta-D-galactosidase (EC 3.2.1.23). 6. Preliminary investigation also indicated that, whereas glucose 6-O-sulphate and glucose 3-O-sulphate were were neither substrate nor inhibitor of glucose oxidase (EC 1.1.3.4), galactose 6-O-sulphate was oxidized half as fast as unsubstituted galactose by galactose dehydrogenase (EC 1.1.1.48).     

Chemical modifications of the crystalline quinolinate phosphoribosyltransferase from hog liver.

Amino acid analysis and chemical modification of the crystalline quinolinate phosphoribosyltransferase (EC 2.4.2.19) from hog liver were performed. The enzyme contained 29 residues of half cystine per mol. The enzyme activity was strongly inhibited by sulfhydryl reagents. The number of reactive (exposed) sulfhydryl group was determined to be 10.2 and total sulfhydryl group was to be 25.2 per mol by using 5,5'-dithiobis(2-nitrobenzoic acid). The enzyme activity was also inhibited by lysine residue-, histidine residue-, and arginine residue-modifying reagents. These results and the effect of preincubation with the substrates on chemical modifications suggest that the lysine residue, histidine residue and sulfhydryl group may be closely related to the binding site of quinolinic acid.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

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.     

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).     

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.     

New insights into the active center of rat liver cystathionase.

Treatment by urea of purified rat liver cystathionase (L-Cystathionine cysteine-lyase (deaminating), EC 4.4.1.1) provoked a similar alteration of two activities of the enzyme, namely cysteine desulfhydration and homoserine deamination. Since the decreases of the two activities were also comparable as a result of chymotrypsin digestion of the enzyme, these observations suggest that the two sites responsible for the one and the other activites are in close proximity. Studies of the effect of derivatives of substrates (S-carboxymethylcyste-ine, S-carboxyethylcysteine, S-carboxymethylhomocysteine and S-carboxyethylhomocysteine) on both activities were performed. All of them inhibited cysteine desulfhydration and homoserine deamination; in several cases, the type of inhibition was also determined. The results are in agreement with the hypothesis that each of the two sites of the active center has, at least, three binding points which "recognise" groupings of substrates or of inhibitors, and this led us to propose a model for the active center. Each site has an -NH-2 binding point, hence the active center has two -NH-2 binding points; therefore, as cystathionase consists of four subunits and contains four molecules of pyriodoxal phosphate, it might be of interest to determine whether the smallest active molecule is the dimer.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

A neutral collagenase from human gastric mucosa.

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins alpha2-macroglobulin and a smaller component of mol.wt. approx. 40000; alpha1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.     

Studies on the cholesterol ester hydrolase of Trogoderma (Coleoptera).

The enzyme cholesterol ester hydrolase (EC 3.1.1.13) was detected in the larvae of the khapra beetle, Trogoderma granarium (Everts). The pH and temperature optima for the enzyme were 6.6 degrees and 37 degrees C respectively. The mol.wt. of the enzyme was 76000-80000. The enzyme was equally effective in hydrolysing cholesteryl acetate, stearate and oleate. Cholesterol derivatives, namely the chloride and the methyl ether, inhibited the enzyme activity almost completely. It was also inhibited completely by p-hydroxymercuribenzoate. This inhibition was reversed by the addition of dithiothreitol, reduced glutathione or cysteine. The enzyme activity was associated predominantly with the 104000 g fraction.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Human beta-glucosidase: inhibition by sulphates and purification by affinity chromatography on Dextran-sulphate-Sepharose

The acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from human placenta is inhibited by sulphated macromolecules such as Dextran sulphate or chondroitin sulphate. This inhibition is alleviated by compounds such as crude taurocholate or phospholipids, which are known activators of acid beta-glucosidase. Partially-purified human beta-glucosidase will bind to Dextran sulphate linked to Sepharose 4B and can be eluted with low concentrations of crude sodium taurocholate. This procedure gives a 10-15 fold purification with good yield and has been included in a scheme giving an approx. 4000-fold purification of placental beta-glucosidase. Evidence is presented which suggests that phospholipids bind to beta-glucosidase by both ionic and hydrophobic interactions. The inhibition of enzyme activity caused by sulphated compounds and non-ionic detergents may be attributed to interference with, respectively, the ionic and hydrophobic binding of phospholipid to the enzyme.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Improved purification and some properties of bovine lysosomal carboxypeptidase B

Lysosomal carboxypeptidase B (peptidyl-L-amino-acid hydrolase, EC 3.4.18.1) from bovine spleen purified to apparent homogeneity was found to have a molecular weight of 52 000 in the absence and of 25 000 in the presence of urea, determined by gel filtration, indicating the existence of two subunits of identical size. The amount of approx. 15% carbohydrate estimated after cleavage by endoglycosidase H was shown to be insignificant for enzymatic activity. The isoelectric focusing separated lysosomal carboxypeptidase B into several protein bands - each enzymatically active - with a range of isoelectric points between 4.6 and 5.2. The titration of the sulphydryl group in the active site of the enzyme with the proteinase inhibitor E-64 yielded one thiol group per molecule. A maximum of activation was achieved by the addition of selenocystamine together with dithioerythritol and EDTA in the incubation solution. Under these conditions the carboxypeptidase hydrolyzed benzoylglycylarginine (80 kat/mol enzyme), benzoylarginine amide (38 kat/mol enzyme) and carbobenzoxyglutaryltyrosine (110 kat/mol enzyme). Slight enzymatic activities towards benzoylarginine 2-naphthylamide and benzoylarginine p-nitroanilide could be measured. With the oxidized insulin B chain, lysosomal carboxypeptidase B exhibited only carboxypeptidase activity.     

Characterization of a new inhibitor for angiotensin converting enzyme from the venom of Vipera aspis aspis

1. Angiotensin converting enzyme inhibitor has been isolated from the venom of Vipera aspis aspis by gel filtration and reverse phase HPLC. 2. The purified inhibitor is a decapeptide, whose amino-terminal is blocked, with mol. wt 1044 determined by fast atom bombardment mass spectrometry. 3. The peptide inhibited the conversion of angiotensin I to angiotensin II, and Ki values were determined to be 7.54 x 10(-4) and 1.36 x 10(-4) M, respectively, using Hip-His-Leu and Hip-Gly-Gly as substrates 4. The peptide also inhibited the degradation of bradykinin, induced hypotension in spontaneously hypertensive rats and caused an increase in capillary permeability in rabbits, however, it possessed no lethality.     

The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.