Purification and properties of trehalase from monkey small intestine

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had aK ^m value of 17.4 mM for trehalose and a V_max of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.     

Purification and partial characterization of dimeric dihydrodiol dehydrogenase from monkey kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of several monkey tissues, among which kidney exhibited the highest activity and contained a high-molecular weight (Mr approximately 65,000) enzyme species. The enzyme species was purified to apparent homogeneity and showed a subunit molecular weight of 39,000. The enzyme oxidized benzene dihydrodiol (Km = 0.9 mM) at a pH optimum of 9.8, and highly reduced vicinal diketones such as camphorquinone (Km = 0.1 mM) and diacetyl (Km = 0.8 mM) around pH 7.5, but alicyclic alcohols, hydroxysteroids and ketosteroids were inactive substrates for this enzyme. Quercitrin, SH-reagents, stilbestrol were inhibitory to the enzyme activity, but other synthetic estrogens, anti-inflammatory agents and 3-ketosteroids were not.     

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.     

Purification and properties of enolase from swine kidney

The first, enolase (2-phospho-d-glycerate hydrolyase, EC 4.2.1.11) to be isolated from a gluconeogenic tissue, swine kidney, was purified more than 600-fold to near homogeneity, as estimated from sedimentation equilibrium and velocity measurements and from disc electrophoresis patterns. The physical properties of the enzyme were examined. Purified kidney enolase has a s^0.87%_20,w = 5.87 S, M_r = 90,000 ± 4,500, e^0.1%_280,1cm = 1.07/mg/ml, a Stokes radius of 37.0 Å, and an apparent subunit molecular weight of 52,000.The amino acid composition was determined and compared with those of mammalian muscle enolases. The partial specific volume calculated from the amino acid composition was found to be 0.728 cc/g. Swine kidney enolase had 12 cysteines per mole; in the native enzyme, two reacted with DTNB.The enzyme was stabilized by magnesium, sucrose, or glycerol; activity lost, on prolonged storage could be completely recovered by treatment with mercaptoethanol and EDTA at 37 °C. Some evidence was obtained for the existence of active monomers of this enzyme. This form of swine kidney enolase was quite unstable, however. The pH optimum was at 6.8. The Michaelis constants for 2-phospho-d-glycerate and phosphoenolpyruvate were 5.10^?5m and 10^?4m; that for magnesium was 4.10^?4m. Substrate inhibition was found for 2-phosphoglycerate but not for phosphoenolpyruvate. No inhibition is seen under comparable conditions with mammalian enolases from glycolytic tissues. This finding is discussed.     

Purification and properties of AMP-deaminase from human kidney.

AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMP-deaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (S0.5) as high as 10 mM. ATP at 1 mM strongly activated the enzyme, decreasing S0.5 nearly 10-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (Ki approximately 16 mM) and had a pure competitive character. At pH 7.2, physiological for the kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each.     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

Purification and properties of pyruvate dehydrogenase kinase from bovine kidney

Pyruvate dehydrogenase kinase was purified about 2,700-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase consists of two subunits (alpha beta) with molecular weights of 48,000 (alpha) and 45,000 (beta) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity resides in the alpha subunit. The alpha subunit is sensitive to proteolysis by chymotrypsin, whereas the beta subunit is selectively modified by trypsin. These observations, together with the results of peptide mapping, indicate that the two subunits are distinctly different proteins. It is proposed that the beta subunit is a regulatory subunit.     

Purification and properties of alpha-aminoadipate aminotransferase from rat kidney.

Previous studies with rat kidney preparations indicated that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.