Immunological relationships of bacterial L-asparaginases.

Rabbit antisera against L-asparaginase preparations from Escherichia coli, Erwinia carotovora, Citrobacter sp. and Chromobacterium violaceum showed on immunoelectrophoresis that only the enzymes from E. coli and Citrobacter are immunologically related. Purified preparations had to be used to determine the immunological cross-reactions. Immunoelectrophoresis at different pH values yielded the zero mobility points of the enzymes. The activity of the Er. carotovora preparation was enhanced up to fourfold by homologous antiserum but not by normal sera. Heterologous antisera also enhanced, but only at a higher concentration. Less enhancement was observed for the other enzymes with antisera as well as with bovine serum albumin. Inhibition was not observed.     

Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum.

The corticosteroid-binding globulin from guinea pig pregnancy serum was purified by the sequential use of affinity chromatography, hydroxylapatite chromatography, and gel filtration chromatography at a cumulative yield of 80%. The protein was found to be homogeneous by analytical gel electrophoresis, equilibrium sedimentation ultracentrifugation, immunoelectrophoresis, and stoichiometry (1:1) of steroid binding. Guinea pig corticosteroid-binding globulin has a molecular weight of 43 300 and contains 29% carbohydrate. The intrinsic fluorescence of the corticosteroid-binding globulin is quenched by about 73% when 1 mol of cortisol is bound. The association constants (pH 7.4) at 4 and 37 degrees C are 2.5 X 10(7) and 1.5 X 10(6) M-1 for cortisol and 1.4 X 10(6) and 0.2 X 10(6) M-1 for progesterone, respectively.     

Purification and properties of guinea pig liver ornithine transcarbamylase]

Ornithine transcarbamylase, the enzyme which catalyzes the formation of citrulline from ornithine and carbamoylphosphate, has been purified from guinea pig liver. By the procedure indicated in the present paper a 200 fold purification of the enzyme has been achieved. Using both the purified fraction and the crude extract, a parallel determination of some physicochemical properties has been carried out. The pH of maximal activity of OTC was 7.8 for both preparations. The maximal stability of the enzyme with respect of pH showed a plateau over the range of pH 7 to 9.5 in the purified fraction, whereas the crude extract exhibited a major stability which lay between pH and 10. Both OTC preparations showed similar behavior regarding thermal stability, the enzyme being still active at a 50 degrees C temperature. The values of the apparent Km's proved to be 4.4 mM for the substrate ornithine and 5 mM for carbamoylphosphate.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

Purification and characterization of guinea pig liver morphine 6-dehydrogenase

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.     

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.     

Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver.

NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.     

17beta-Hydroxy-C19-steroid dehydrogenases from male guinea pig liver. Purification and characterization

One major and six minor 17beta-hydroxy-C19-steroid dehydrogenases were isolated each in a highly pure form from male adult guinea pig liver by a combination of gel filtration and ion exchange chromatography. Molecular weight, amino acid composition, sugar content, number of sulfhydryl groups. NH2-terminal amino acid, isoelectric point, substrate specificity, pH optima, Km values, and inhibitory effect of other steroids were studied. The amino acid composition, Km values, and substrate specificity indicated two separate groups of enzymes. The first group possessed a dual coenzyme requirement and specificity for 5beta-androstanes, whereas the second group showed apparent TPN+ and 5alpha-androstane specificities. Phosphate enhanced the DPN+-related activity of the first group and evoked DPN+-linked activity in the second group of enzymes. A molecular weight of 31,000 to 32,000 with a single chain structure was estimated for four of the enzymes. The other three enzymes consisted of two components of 24,000 and 11,000 daltons.     

Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver.

Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.     

Kinetic and inhibition studies on reduction of diphenyl sulfoxide by guinea pig liver aldehyde oxidase

To characterize the properties of diphenyl sulfoxide (DPSO) as a new type of electron acceptor for guinea pig liver aldehyde oxidase (AO), we compared the kinetics of the reductions of DPSO and other classical electron acceptors such as O2 and ferricyanide. The double-reciprocal plot of the 2-hydroxypyrimidine (2-OH PM)-linked DPSO reduction with the highly purified enzyme was biphasic. Similar biphasic plots were obtained with the reductions of other electron acceptors. Only the lower Km value, which was obtained by extrapolation of the plot at lower concentrations of 2-OH PM, was identical with that shown by the freshly prepared crude enzyme. DPSO as well as menadione progressively inhibited the reductions of O2 and ferricyanide with time. Menadione inhibited the DPSO reduction noncompetitively with respect to 2-OH PM and competitively with respect to DPSO, while its mode of inhibition of ferricyanide reduction was uncompetitive for either the electron donor or the acceptor. On the other hand, DPSO showed an uncompetitive and a noncompetitive inhibition of ferricyanide reduction with respect to 2-OH PM and ferricyanide, respectively. These results may indicate that DPSO interacts with the enzyme at the same site as menadione, and thereby when other electron acceptors are present it serves as an actual inhibitor rather than as an electron acceptor for AO.