Substrate specificity and kinetic properties of neutral maltase of human granulocytes

A neutral maltase immunologically similar to this of kidney exist in human granulocytes. We have studied some kinetic properties of this enzyme on a microsomal fraction of granulocytes. Its optimal pH is very closed of 6.8 and this enzyme, highly specific for maltose, hydrolysis very weakly the nigeriosis. Maltotriose, maltotetraose and maltopentanose are inhibitors of this enzyme, which is not inhibited by all disaccharides studied.     

Comparative study of the kinetic properties of the neutral maltase of human granulocytes and of the kidney maltase of the rat

Neutral maltase from human granulocytes has a different substrate specificity from the human neutral maltase of kidney, though it has been reported that these two enzymes are immunologically similar. We report here that human granulocyte neutral maltase is similar to the neutral maltase from rat's kidney as regards the substrate specificity and the inhibition by Tris and maltodextrins. We also report a different thermal stability that might imply some structural differences between the two enzymes.     

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 some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Neutral maltase of human granulocytes: localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.     

Effect of neutral proteases from human granulocytes on colony forming cells in vitro.

Marked inhibition of colony formation is observed after incubation of mouse and human bone marrow cells with the human granulocytic neutral proteases elastase and chymotrypsin as well as with pancreatic chymotrypsin. The corresponding enzymes inactivated with diisopropylfluorophosphate were almost inactive. Incubation of different colony inducing agents either resulted in no change or in an increase of their colony stimulating activity. The data suggest a direct proteolytic action of the proteases on colony forming cells which may alter receptor sites for colony stimulating activities.     

Purification and properties of gamma-glutamylcyclotransferase from human erythrocytes.

1. GAMMA-Glutamylcyclotransferase was purified 10000-fold from human erythrocytes. 2. The purification steps involved fractionation with (NH4)(2)SO(4) and chromatography on Sephadex G-75, DEAE-cellulose and hydroxyapatite. The purified enzyme was found to be homogeneous on density-gradient polyacrylamide-gel electrophoresis. 3. The maximum reaction rate was observed at pH9.0 and the apparent Km value for gamma-glutamyl-L-alanine was 2.2mM. 4. The molecular weight (25250) of the purified enzyme agreed well with the value (25500) in fresh haemolysates, indicating no apparent structural modification of the enzyme during purification. However, rapid processing of the blood through the initial (NH4)(2)SO(4) and Sephadex-chromatography steps was required to prevent formation of a high-molecular-weight aggregate with substantially lower specific activity. 5. gamma-Glutamylcyclotransferase catalyses the formation of 5-oxoproline from gamma-glutamyl dipeptides. The role of this enzyme in erythrocytes is of particular interest, because gamma-glutamyl-L-cysteine serves as a substrate for both gamma-glutamylcyclotransferase and glutathione synthetase. Thus the cyclotransferase could modulate glutathione synthesis.     

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.     

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.     

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.     

Purification of granulocyte neutral protease from human blood and rheumatoid synovial fluid.

The neutral protease activity of human synovial fluid cells, like that of peripheral blood leucocytes, is located in a granule fraction. It can be solubilised by various agents but only 1 M neutral salts do so without inactivation. Salt-solubilised neutral protease has been purified (300 X) from synovial fluid cells; like preparations obtained in the same way (600 X purified) from peripheral blood leucocytes, it has a broad pH profile of activity (pH 7--10.5) and in this, as well as in substrate specificity and sensitivity to activators and inhibitors, it behaves as a serine-histidine type protease similar to elastase (EC 3.4.21.11). The product showed two major components on polyacrylamide gel electrophoresis. Collagenase or chymotrypsin-like activity were not detected.     

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.