Affinity chromatography and some properties of the angiotensin-converting enzyme from human heart

Human heart angiotensin-converting enzyme has been purified by affinity chromatography on immobilized N-[1(S)-carboxy-5-aminopentyl]-Gly-Gly. The isolation procedure permitted a 1650-fold-purified enzyme to be obtained. The specific activity of angiotensin-converting enzyme was 38 units per mg protein. The molecular weight of enzyme determined by polyacrylamide gel electrophoresis in denaturing conditions was 150,000. The isoelectric point (5.3) of the enzyme was determined by chromatofocusing. The Km values of the enzyme for Bz-Gly-His-Leu and angiotensin I are 1.2 mM and 10 microM, respectively. Substrate inhibition of heart angiotensin-converting enzyme with a K's of 14 mM has been shown. The human heart enzyme is inhibited by SQ 20881 (IC50 = 40 nM). It was shown that NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- are activators of the heart angiotensin-converting enzyme, whereas CH3COONa and NaNO3 have no effect on a catalytic activity of the enzyme.     

Purification and study of the physicochemical properties of angiotensin converting enzyme from the human liver

Angiotensin-converting enzyme (ACE) from human liver was first purified 9000-fold by chromatofocusing with 22% yield. The enzyme had a specific activity of 10 U/mg. The enzyme molecular weight was 150000, as determined by electrophoresis in a 7.5% polyacrylamide gel. The enzyme pI determined by chromatofocusing was 4.2-4.3. KM of human liver ACE, measured using hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine as substrates, was 5 mM and 0.1 mM, respectively. Human liver ACE was inhibited by SQ 20881 with IC50 equal to 1.8 X 10(-8) M.     

Isolation and properties of the angiotensin-converting enzyme from human lungs

Using chromatofocusing, an angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human lung. The procedure allows for 24 300-fold purification of the enzyme. The enzyme specific activity is 36.3 u. per mg protein; Mr as determined by polyacrylamide gel electrophoresis is 150 000. The lung enzyme after solubilization by trypsin treatment was found to be heterogeneous. Four isoforms of the enzyme with pI 5.3, 4.9, 4.8 and 4.6 were identified. The pH-optimum for the enzyme with respect to hippuryl-L-histidyl-L-leucine hydrolysis lies at 8.3; Km = 2.8 mM. The effect of Cl- on the enzyme activity was studied. It was found that the bradykinin-potentiating factor (SQ 20 881) inhibits the human lung angiotensin-converting enzyme (I50 = 1.6 X 10(-8) M).     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

The purification and properties of ox liver short-chain acyl-CoA dehydrogenase.

The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.     

Atriopeptin 2 is hydrolysed by cardiac but not pulmonary isozyme of angiotensin-converting enzyme

Hydrolysis of Bz-Gly-Ser-Phe-Arg, C-terminal fragment of atriopeptin 2, by human cardiac angiotensin-converting enzyme has been studied. The KM for the reaction was 10(-4) M. The effect of concentration of NaCl on activity of cardiac angiotensin-converting enzyme has been determined, which allowed to regard Bz-Gly-Ser-Phe-Arg as bradykinin-like substrates. It was demonstrated that cardiac, but not pulmonary isozyme of angiotensin-converting enzyme specifically hydrolyses atriopeptin 2.     

Various properties of angiotensin-converting enzyme from the seal Phoca groenlandica

It was found that the molecular mass of the angiotensin-converting enzyme from seal (Phoca groenlandica) lungs determined by electrophoresis in 7.5% PAAG in the presence of sodium dodecyl sulfate is 150 kD. The enzyme has a pH optimum with respect to hippuryl-L-histidyl-L-leucine at 7.3--7.5; KM is 1.2 mM. The enzyme is inhibited by the substrate to form a nonproductive ES2 complex with the dissociation constant (Ks') of 4.8 mM. The activation of the seal angiotensin-converting enzyme in the presence of NaCl was studied. The bradykinin-potentiating factor (SQ 20881) inhibits the seal enzyme with a high efficiency (IC50 = 2.5.10(-8) M). Monoclonal antibodies to the angiotensin-converting enzyme from human lungs do not interact with its seal lung counterpart, which points to the species specificity of the angiotensin-converting enzyme.     

Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.     

Purification and characterization of human hepatic cysteine-conjugate beta-lyase.

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5'-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.     

Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties.

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.     

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

人脑醛糖还原酶的纯化与性质

联合采用ＤＥＡＥ－纤维素层析、色谱聚焦、ＮＡＤＰ亲和层析与ＳｅｐｈａｄｅｘＧ－１００的凝胶过滤,对人脑醛糖还原酶（ＥＣ１．１．１．２１;ＡＬＲ）进行纯化．现测得该酶的等电点ｐＨ值为５．８５．经聚丙烯酰胺凝胶盘状电泳和Ｗｅｓｔｅｒｎ印迹证实,获得了满意的酶纯度．同葡萄糖,葡糖－６－磷酸与ＮＡＤＰＨ保温后,人脑ＡＬＲ纯品的活性与对照酶组相似,且不被糖酵解途径的一些磷酸化中间产物抑制．苯基硼酸琼脂糖柱层析洗脱谱峰和氢硼化钠还原反应提示,当同葡萄糖保温时,人脑ＡＬＲ（特别是其均一态）可能未被进一步糖基化．在糖尿病并发症和按结构完成药物设计的研究工作中,纯品ＡＬＲ的应用可发挥重要作用     

Rapid affinity chromatographic purification of human lung and kidney angiotensin-converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1(S)-carboxy-5-aminopentyl]glycylglycine

Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly (N-[1(S)-carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, Ki = 0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160,000. The Km with respect to hippuryl-L-histidyl-L-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.     

Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.     

Partial separation of allelic forms of N-acetyltransferase from the rabbit liver using a new method of enzyme purification

A new effective method for purification of rabbit liver N-acetyltransferase to apparent homogeneity has been developed. The method consists in polymin P and ammonium sulfate fractionation, DEAE-Sephacel and Ultragel AcA 44 chromatography and chromatofocusing. In final preparations the enzyme was purified 2500-3000-fold and its specific activity was found to be about 3000-4000 units per mg of protein. During chromatofocusing of enzyme preparations on a middle pressure chromatograph FPLC (Pharmacia, Sweden) a partial separation of acetyltransferase allelic forms from fast and slow-acetylators took place. The supposed allelic acetyltransferase forms differ in some biochemical properties. In particular, the slow acetyltransferase form is much more sensitive towards 0.1 M KCl against the rapid enzyme form. It is assumed that the differences between the catalytic properties of acetyltransferase from rapid and slow acetylators may be explained by differences between their polypeptide primary structures.     

Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae

Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.