Effect of different compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.     

Measurement of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase activity in human tissues.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.     

Purification and some properties of gamma-glutamyltransferase from human liver.

The purification of gamma-glutamyltransferase ((gamma-glutamyl)-peptide: amino acid gamma-glutamyltransferase, EC 2.3.2.2) from normal human liver is described. The procedure includes solubilization of enzyme from membranes using deoxycholate and Lubrol W, treatment with acetone and butanol, and affinity chromatography on immobilized concanavalin A. Treatment with papain was used to release the enzyme from aggregates of lipid and protein, prior to further purification. On overall purification of 9400 was achieved and analytical polyacrylamide gel electrophoresis indicated that the final product was homogeneous, and had a molecular weight of 110 000. Two subunits were identified on dodecyl sulfate gel electrophoresis with estimated molecular weights of 47 000 and 22 000. The kinetic properties studied for the purified enzyme were similar to those found for partially purified (not papain-treated) enzyme, and resembled those of serum gamma-glutamyltransferase. The true KM values for the liver enzyme were estimated to 0.81 mM for gamma-glutamyl-p-nitroanilide and to 12.4 mM for glycyl-glycine.     

Purification and properties of 5,6-dihydropyrimidine amidohydrolase from calf liver

5,6-Dihydropyrimidine amidohydrolase was isolated from an acetone powder of calf liver and purified to homogeneity. Purification made use of heat treatment, ammonium sulfate fractionation and chromatography on Chelating Sepharose and DEAE-Sepharose with 44% recovery of total activity. The native enzyme has a molecular mass of 217 kDa consisting of four subunits with a molecular mass of 54 kDa each. The amidohydrolase is a metalloenzyme containing one zinc atom/subunit. The enzyme can slowly be inactivated by chelating agents. The kinetic parameters for substrates, 5,6-dihydrouracil, 5,6-dihydrothymine and glutarimide were determined. From log Vmax/KM data, a pKa of 7.6 could be calculated suggesting the formation of a zinc-bound hydroxyl ion which carries out the nucleophilic attack on the C-4 of dihydrouracil.     

N-氨甲酰水解酶的纯化及性质

从Burkholderiacepecianjut1分离纯化N氨甲酰D氨基酸水解酶(NDase)。实验表明,该酶亚基35KD,最适温度为52℃,最适pH为7.2左右。以N氨甲酰D苯丙氨酸作底物,其米氏常数Km为10.22mmol/L,最大反应速度Vmax为0.27mmol/(L·min)。实验表明二价金属离子对酶活有重要影响。     

Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida.

Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.     

N-Acetylaminoacyl-p-nitranilidase from human placenta. Purification and some properties.

An enzyme hydrolyzing N-acetylaminoacyl-p-nitroanilides has been isolated from mature human placentae by a six-step procedure comprising extraction from a placenta homogenate, ammonium sulfate fractionation, treatment with isopentyl alcohol, chromatography on CM-Sephadex, protamine sulfate precipitation and gel filtration on an Ultrogel AcA 34 column. About 2500-fold enrichement was achieved from placenta homogenate. The purified enzyme preparation showed a single band on polyacrylamide disc electrophoresis. The molecular weight was estimated to be 380,000 by gel filtration. Placental extracts contain two main isoenzymes of pI 3.9 and 4.5 respectively. Activity was strongly inhibited by chloromercuribenzoate, slightly inhibited by Ca2+ and cysteine; no activation could be detected. The enzyme exhibits an exopeptidase-like activity towards acetyl-dipeptides with a certain specifity towards N-acetylalanyl-alanine; N-acetylalanine-p-nitroanilide, however, is hydrolyzed four times faster. With N-acetylalanine-p-nitroanilide as substrate the pH optimum was 8.0--8.2; Km was 2.13 mmol/l. N-Acetylleucine-p-nitroanilide and N-acetyltyrosine-p-nitroanilide were split slowly; N-acetylalanyl-alanyl-alanine-p-nitroanilide, N-butyloxycarbonyl-alanyl-alanine-p-nitroanilide, unsubstituted analogous aminoacyl-p-nitroanilides and several protein substrates were not hydrolyzed. The functions of the enzyme are still unknown.     

Purification and some properties of arginase from human lung.

Arginase from human lung has been isolated and purified about 100-fold. During the purification procedure the enzyme was stabilized by Mn2+. The molecular weight determined by Sephadex G-150 gel filtration was found to be 120 000. The enzyme is highly specific towards L-arginine. Incubation of the enzyme with EDTA for 60 min at pH 7.5 and 37 degrees C results in dissociation into inactive subunits of mol. wt. 30 000. On addition of Mn2+ ion to the inactivated enzyme, the subunits reassociate into the native form of the enzyme of mol. wt. 120 000, and the activity is restored.     

Purification and properties of cystathionine synthase from human liver.

Cystathionine β-synthase has been isolated from human liver in two enzymically active forms. Both enzymes, α and β possess a molecular weight of 250,000 and are dependent upon pyridoxal phosphate as a cofactor.     

Purification and properties of neutral beta-galactosidases from human liver.

The synthesis of collagen under conditions in which polypeptide chain initiation is selectively inhibited by medium hypertonicity was compared to the synthesis of other proteins in chick embryo leg bone cells in monolayer cultures. Three different approaches showed that collagen synthesis is far more sensitive than the majority of other cellular proteins to the hypertonic initiation block. In marked contrast, the synthesis of an unidentified protein, migrating with an apparent molecular of 45,000 to 50,000 is particularly resistant to hypertonicity. The effects of hypertonic conditions were found to be readily reversible upon restoration of isotonicity. Since these suboptimal growth conditions can decrease the amount of collagen synthesized relative to total protein synthesis, they provide an experimental model for the study of the translational control of the synthesis of collagen and other proteins.     

Purification and properties of N-carbamylputrescine amidohydrolase from maize shoots

N-Carbamylputrescine (NCP) amidohydrolase was purified ca 70-fold from maize shoots. The enzyme was present in the cytosol and the optimal pH w     

Crystallization and some properties of acetylpolyamine amidohydrolase from Mycoplana bullata

During the course of investigations on the catabolism of acetylpolyamines by microorganisms, we found that acetylpolyamine amidohydrolase was produced by Mycoplana bullata FERM BP-1845 and isolated the enzyme from the cell-free extract in crystalline form. The enzyme had an apparent molecular weight of 67 kDa and was composed of two identical subunits. The enzyme activity was inhibited by o-oxyquinoline and the crystalline enzyme contained one zinc atom per each subunit. The enzyme had an optimal pH around 8.0 with acetylputrescine as substrate and showed broad substrate specificity and high affinity towards various acetylpolyamines, such as acetylputrescine, acetylcadaverine, acetylspermidine, and acetylspermine.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.