Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Inorganic pyrophosphate: fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli

A procedure was developed for the purification of inorganic pyrophosphate: fructose-6-phosphate 1-phospho-transferase (PPi-PFK) from potato tubers. The enzyme has the structure alpha 4 beta 4 with a subunit of 68 kDa and a beta subunit of 60 kDa. The structural relationship of this enzyme to other PFKs and to fructose bisphosphatase was examined by immunoprecipitation and immunoblotting. Antibodies to the plant enzyme did not react with E. coli PFK. No cross-reaction was seen among the following enzymes or their antibodies: yeast fructose bisphosphatase; rabbit PFKs A, B, or the enzyme from brain; and the two subunits of the potato PPi-PFK. On the other hand, antibody to E. coli PFK-1 strongly cross-reacts with the 60 kDa polypeptide but not 68 kDa peptide.     

Molecular characterisation of a novel thermophilic nitrile hydratase

The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases.     

High density lipoprotein exchange reactions

The Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein. The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively. According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested. The catalytic constants were found to be higher at pH = 7.6 than at pH 10.0 for all the three enzymes. This relative increase was much more pronounced in the case of the porcine enzyme. The KM value at pH = 10.0 was also significantly higher for the porcine enzyme. Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.     

cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.     

Mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase

Methods for mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase are described with a view to using such data for fine-structure analysis of mutations and/or modifications affecting the protein's electrostatic charge. Peptides generated from [35S]methionine-labeled regulatory subunit were separated by high-resolution two-dimensional gel electrophoresis. Sites of papain cleavage in denatured regulatory subunit were deduced from the kinetics of the appearance, molecular weights, and relative isoelectric points of the fragments produced. These sites and sites of chymotrypsin digestion in the native protein were confirmed by studying peptide overlaps. Carboxy-terminal peptides were identified both by overlaps with cyclic AMP-binding chymotryptic fragments and by their preferential labeling during polysome runoff mediated by pactamycin, an inhibitor of protein initiation. Since peptides containing modifications or mutations that alter protein charge can be identified by shifts in first-dimension isoelectric focusing gel positions, knowledge of fragment endpoints will permit rapid mapping of sites of such alterations by two-dimensional gel analysis of partial proteolytic digests. Such a mapping procedure is inexpensive, can be applied to partially purified proteins or to proteins eluted from polyacrylamide gels, requires only nanogram amounts of the protein of interest, and does not require sequence data to determine relative positions of peptides. Therefore, it provides an attractive alternative to more classical peptide analysis for studying point mutations in cellular proteins of low abundance.     

Purification,substrate specificity,and N-terminal amino acid sequence analysis of a β-lactamase-free penicillin amidase from <Emphasis Type="Italic">Alcaligenes</Emphasis> sp.

A -lactamase-free penicillin amidase from Alcaligenes sp. active against various -lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.     

Purification and partial amino acid sequence of papain-solubilized class II transplantation antigens

Papain-solubilized human class II (HLA-DR) antigens have been purified from cadaveric spleens by ion-exchange chromatography, gel chromatography, and immunosorbent purification. The isolated papain-solubilized antigens comprised two subunits with apparent molecular weights of 23 000 and 30 000, respectively. The circular dichroism spectrum for the isolated class II antigens was similar to spectra recorded for HLA-A, -B, and -C antigens, immunoglobulins, and immunoglobulin fragments. Thus, class II antigens contain a considerable amount of beta structure. The small subunit (beta chain) exhibited extensive charge heterogeneity on two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, whereas the large subunit (alpha chain) was more homogeneous. The structural heterogeneity of beta chains remained after neuraminidase treatment. The NH2-terminal amino acid sequence of the beta chains displayed multiple residues in several positions in accordance with the genetic polymorphism displayed by this chain. The alpha chain also displayed multiple residues in some positions, suggesting either that some of the genetic polymorphism of the class II antigens may be endowed in this chain or that multiple loci control the expression of several alpha chains. Papain-solubilized class II antigen subunits were homologous in their amino acid sequences with HLA-DR antigens of defined antigenic specificity as well as with murine I-E/C antigens.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.     

Glutathione S-transferase in the insect Apis mellifera macedonica kinetic characteristics and effect of stress on the expression of GST isoenzymes in the adult worker bee

The glutathione S-transferase present in the adult worker bee Apis mellifera macedonica was purified and analyzed for its physicochemical and kinetic properties. The enzyme is heterodimeric with subunit molecular masses of 29 and 25 kDa, respectively. Two main isoenzymes with distinct kinetic properties are present, with isoelectric points of 7.40 for the alkaline and 4.58 for the acidic forms, respectively. The two enzymes are induced independently by factors such as insecticide treatments and environmental conditions, including low temperatures or starvation.     

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.     

Purification and characterization of trimming glucosidase I from pig liver

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.     

Isolation and characterization of dihydropteridine reductase from human liver.

Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.     

Effects of charged water-soluble polymers on the stability and activity of yeast alcohol dehydrogenase and subtilisin Carlsberg.

Remarkable increases in enzyme catalytic stability resulting from addition of charged water-soluble polymers have recently been reported, suggesting that use of these polymers may be an attractive general strategy for enzyme stabilization. To test the proposed hypothesis that coulombic forces between water-soluble polymers and enzymes are primarily responsible for enzyme stabilization, we examined the catalytic stability and activity of two enzymes in the presence of polymers differing in net charge. All polymers tested increased enzyme lifetimes, regardless of their net charge, suggesting that stabilization of these enzymes by water-soluble polymers is not solely dependent on simple electrostatic interactions between the polymers and enzymes.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Purification and physical and kinetic characterization of an NAD+-dependent malate dehydrogenase from leaves of pineapple (Ananas comosus)

An NAD⁺-dependent malate dehydrogenase (MDH, EC 1.1.1.37) was purified and characterized from leaves of pineapple ( Ananas comosus ), a plant with Crassulacean acid metabolism (CAM). The purified enzyme had a subunit molecular mass of 39.5 kDa. Its activity showed a maximum at pH 6.8–7.0 and decreased sharply towards pH 8.0. This activity profile coincided with a change in the aggregation state, as determined by gel filtration on high-performance liquid chromatography from a dimer at pH 7.0 to a tetramer at pH 8.0. This isozyme is one of at least 5 MDH in pineapple leaves distinguishable by non-denaturing isoelectric focusing and displayed an isoelectric point of 5.8. The ratio of oxaloacetate reduction versus malate oxidation rates varied between 431 and 52 at pH 6.8 and 7.5, respectively. Antibodies raised against the purified pineapple leaf MDH immunodecorated a single 39.5-kDa polypeptide in denatured crude leaf extracts, but did not cross-react with extracts from purified pineapple mitochondria possessing high MDH activity. The purified MDH was recognized by monoclonal antibodies raised against the cytosolic MDH from Echinococcus granulosus . These and other distinctive traits, such as its isoelectric point and its subunit mass, suggest that the purified isozyme is the cytosolic MDH. Its properties are consistent with an implied function in the night acidification typical of CAM plants, although it is less clear if it also has a role in the daytime decarboxylation of malate.     

Low molecular weight thermostable β-D-glucosidase from Acidothermus cellulolyticus

Summary Industrial use of cellulase enzymes, including -D-glucosidases, can be greatly improved and expanded by the discovery and characterization of more robust, thermotolerant enzymes. A new thermostable -D-glucosidase was isolated from Acidothermus cellulolyticus culture broth. This enzyme has a molecular weight of 52.5 kDa, an isoelectric pI of 4.1, and a pH activity optimum of 4.5. The optimum temperature for activity on p-nitrophenyl--D-glucoside is 63°C. This value is approximately 20°C higher than temperature optima displayed by most previously characterized -D-glucosidases.     

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) or any other metal cofactor of those tested. The enzyme cleaves specifically (1-->6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM EDTA, while (1-->6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.