Comparative studies on six blood serum glycosidases from several mammalian species.

1. Peripheral blood serum alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, beta-D-xylosidase and beta-D-glucuronidase have been studied with a comparative point of view from several mammalian species: Bos taurus L. (bull), Capra hircus L. (goat), Sus scropha var. domestica L. (pig) and man. 2. Fluorimetric and spectrophotometric procedures were used for determination of enzyme activities and pH optima. 3. Glycosidase activity was generally higher with fluorescent substrates than with chromogenic substrates. 4. alpha-D-mannosidase was the most active with both fluorescent and chromogenic substrates. 5. All the studied enzymes had the same pH optimum (4.0) when the chromogenic substrates were used. 6. pH optima of these glycosidases ranged from 3.0 to 5.5 when the fluorescent substrates were used.     

Acid glycosidases of normal cattle lymphocytes and in chronic lympholeukemia

The activity of five acid glycosidases was determined in lymphocytes from normal animals and animals with chronic lymphocytic leukemia. It was shown that alpha-D-mannosidase activity in leukemic lymphocytes was 5 times lower (p less than less than 0.001) and alpha-D-glucosidase activity was 2 times lower (p less than 0.01) than in normal controls. The progress of the disease and the increase in leukocyte count were accompanied by the decrease in alpha-D-mannosidase activity. No differences have been found in alpha-D-mannosidase properties (thermostability, Km values, ZnSl2 activation) in normal and leukemic lymphocytes.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Properties of acylase preparations from an actinomycete culture

A comparative study of some physico-chemical properties of high-purified preparations of extracellular penicillin-V-acylase and aminoacylase, isolated from the actinomycete Streptoverticillium No 62, revealed the difference in pH and temperature optima, in the sensitivity to the ionic composition of buffer solutions, in the enzyme stability during storage. As for the aminoacylase preparation, its thermostability was studied at different pH values, as well as the effect of specific compounds was tested. Similar to other fungal enzymes, the aminoacylase possesses a wide substrate specificity, and by its stereospecificity can be related to L-aminoacylases, while penicillin-V-acylase is a high-specific enzyme, active against phenoxymethylpenicillin.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.     

Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils

Fluorogenic artificial substrates facilitate sensitive enzyme activity measurements for a variety of processes in soil and other environmental samples. It is possible to use in situ pH for measurements on condition that the substrates are chemically stable. We studied the stability of 12 different methyl umbellipherone (MUF) and amino methyl coumarine (AMC) derivatives used as substrates for arylsulphatase, alpha-glucosidase, beta-glucosidase, beta-xylosidase, cellobiosidase, chitinase, phosphomonoesterase (PME), phoshodiesterase (PDE), esterase, lipase and alanine- and leucine aminopeptidases (AP) over the pH range from 4.0 to 8.0 in modified universal buffer (MUB). Stability of the substrates for lipase (4-MUF-heptanoate) and esterase (4-MUF-acetate) measurements was poor, especially at the higher pH values. Chitinase substrate, 4-MUF-N-acetyl-beta-D-glucosamide, was unstable at high pH values whereas the substrate for PME activity measurement (4-MUF-phosphate) disintegrated at low pH. The other substrates and MUF and AMC standard solutions were stable over the pH range studied. The optima between pH 4 and 8 of the 11 different enzyme activities were measured in three forest and two agricultural soil samples and in one activated sludge sample. In soil, for alanine and leucine AP the pH optima were usually 7.5 or higher, for arylsulphatase, beta-glucosidase, beta-xylosidase, esterase and PDE between 4 and 5.5, and for cellobiosidase between 4 and 5. alpha-Glucosidase had an optimum below 5.5 but also exhibited high activity at pH 7. Soil-dependent variation in pH optima were observed for chitinase, esterase, PDE and PME. Enzyme activities were also measured in 0.5 M acetate buffer at pH 5.5. This buffer yielded the highest activities in all soil samples for arylsulphatase, PDE and PME.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells.

KB cells were synchronized by a double thymidine block procedure. An investigation was made of the activities of alpha-L-fucosidase (EC 3.2.1.51), alpha-D-galactosidase (EC 3.2.1.22), beta-D-galactosidase (ec 3.2.1.23), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), alpha-D-mannosidase (EC 3.2.1.24), beta-D-N-acetylgalactosaminidase (EC 3.2.1.53), and beta-D-N-acetylglucosaminidase (EC 3.2.1.52) from synchronized cultures, using appropriate artificial substrates. Ceramide glucosidase (EC 3.2.1.45) and ceramide trihexosidase levels (EC 3.2.1.47) were also investigated at various stages in the cell cycle, using appropriate glycosphingolipid substrates. Whereas each of these enzymes exhibited some activity throughout the cell cycle, peak activity (2- to 6-fold increase) occurred late in the S phase. Two molecular forms of ceramide glucosidase (optimal activity at pH 4.0 and pH 6.0) and two forms of ceramide trihexosidase (pH 4.0 and pH 7.5) were identified. Peak levels of the forms that preferred the relatively acid pH occurred earlier in the S phase of the cell cycle than those of the forms that were more active at the higher pH. The possibility that the forms with optimal activity at pH 4 are precursors of those with optimal activity at pH 6 to 7.5 is discussed. Precipitation of beta-galactosidase of synchronized KB cells with specific antibody revealed that changes in the activity of this enzyme during the cell cycle were the result of fluctuations in the amount of the enzyme.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Altered glycosidase activity in the liver of rats with galactosamine-induced alpha 1-antiprotease deficiency

Previous studies have shown that chronic administration of D-galactosamine (GalNH2) in rats produces alpha 1-antiprotease (AAP) deficiency and causes accumulation of aberrantly glycosylated AAP in hepatic granules. In order to examine the disordered mechanism which produces this altered glycosylation, the activities of 6 glycosidases in liver homogenates of control and AAP-deficient rats were determined. GalNH2 treatment increases acid pH glycosidase activity, while it decreases intermediate pH alpha-mannosidase and alpha-glucosidase activities. beta-D-Glucosidase, beta-D-mannosidase and beta-D-N-acetylglucosaminidase activities, measured at acid pH, increase more than 2-fold in the GalNH2-treated rats compared to controls. alpha-D-Glucosidase activity measured at intermediate pH decreases 2.5-fold in the experimental rats. alpha-L-Fucosidase and acid phosphatase activities are not significantly changed by GalNH2 treatment. alpha-D-Mannosidase activity can be separated into 2 fractions by ion exchange chromatography. Acid pH alpha-D-mannosidase is increased nearly 2-fold in the GalNH2-treated rats. Intermediate pH alpha-D-mannosidase optimum is decreased alpha-D-mannosidase activities have been observed in humans with AAP deficiency. alpha-Glucosidases and alpha-mannosidases play a crucial role in glycoprotein synthesis. The altered synthesis and structure of AAP in GalNH2-induced AAP deficiency may be a reflection of altered enzyme activities.     

L-Ascorbic acid and lysosomal acid hydrolase activities of guinea pig liver and brain

The effects of L-ascorbic acid deficiency on guinea pig hepatic and brain lysosomal hydrolases were examined. In general, hepatic beta-N-acetylhexosaminidase, beta-D-glucoronidase, alpha-D-galactosidase, alpha-D-mannosidase, and acid phosphatase were elevated in scorbutic animals. This appears to be independent of the starved state. Brain beta-D-glucoronidase and acid phosphatase followed a similar pattern to that observed with the liver enzymes, but brain beta-N-acetylhexosaminidase was not affected by L-ascorbic acid decreased the activity of hepatic beta-N-acetylhexosaminiadase was unaffected by dietary treatments although the activity of beta-N-acetylhexosaminidase A tended to increase in the scorbutic animals. Subcellular fractions were obtained from the three groups of animals and the recoveries of protein, beta-N-acetylhexosaminidase, and glucose-6-phosphatase estimated.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.     

Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) has been purified from the microsomal fraction of pig liver, using an immunoaffinity chromatography, and its properties compared with those of the enzyme purified from pig kidney. The amino acid compositions of both enzymes were similar. The same kinds of carbohydrates were found in both enzymes, but there were differences in the molar concentrations of individual sugars. The liver enzyme had greater concentrations of mannose, fucose and sialic acid than the kidney enzyme, while the concentrations of galactose and glucosamine were greater in the kidney enzyme. The carbohydrates accounted for approx. 18.3 and 22.7% of the weight of the kidney and liver enzymes, respectively. The pH optima, molecular weights, substrate specificities and Km values of the two enzymes and the effects of diisopropylfluorophosphate on their activities were nearly identical. The liver enzyme was heat- and pH-sensitive, but not attacked by proteinases.     

Alterations in glycohydrolase activities in streptozotocin-diabetic Chinese hamsters (Cricetulus griseus).

1. Six weeks after the injection of streptozotocin at 125 mg/kg i.p. in the AV line nondiabetic Chinese hamsters, the animals showed hyperglycemia, increased kidney, pancreas and stomach weights and stomach glucagon contents and depletion of insulin and glucagon in the pancreas. 2. Plasma beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase were elevated; whereas alpha-D-glucosidase was decreased and alpha-D-galactosidase remained unchanged in the plasma. 3. In the kidney, streptozotocin-diabetes led to depression of alpha-D-mannosidase, beta-D-fucosidase and N-acetyl-beta-D-glucosaminidase activities in both 12,000 g supernatant and precipitate fractions, decreases in alpha-D-glucosidase in the supernatant only and no change in alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase and beta-D-glucuronidase. 4. In the liver, significant increases in N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-fucosidase, beta-D-glucosidase and alpha-D-mannosidase were found in either the supernatant or the precipitate fraction of the diabetic animals. The data indicate diabetes-dependent tissue-specific changes in glycohydrolases in the Chinese hamster.     

Purification and characterization of two peroxidases from hard wheat]

Two peroxidases A and B were purified from a borate buffer extract (pH = 10,4) of durum wheat semolina (Triticum durum), var. Bidi 17, by chromatography on DEAE-cellulose, salting out by 3M ammonium sulphate and two chromatographies on CM-cellulose; specific activities of peroxidase A or B were increased 114 or 66 fold. Molecular weight, amino acid composition, absorption spectrum, pH optimum, thermal stability and KM values differentiate the two enzymes. Ion Ca++ was shown as an activator of both peroxidase activities; the presence of an inhibitor in the crude extract was demonstrated.     

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, and alpha-L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of alpha-D-glucosidase to 70% of beta-D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of beta-D-galactosidase to 89% of alpha-D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of beta-D-galactosidase to 85% of alpha-D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.     

Lysosomal membrane adenosine triphosphatase; solubilization and partial characterization

Membranes prepared from Triton WR-1339-filled lysosomes (tritosomes) contained ATPase activity with a pH optimum of 5–8. These membranes also showed adenosine diphosphatase, adenosine monophosphatase, acid β-glycerol phosphatase, and acid pyrophosphatase activities. The soluble (nonmembrane) fraction of the tritosomes also contained these activities, but the properties of the soluble adenine nucleotide phosphatase activities were different from the membrane-associated enzymes. The pH optimum of tritosomal membrane ATPase changed to 5 after solubilization with Triton X-100, but ADPase and AMPase optima remained at 6–7. The pH optimum of intact membrane ATPase was also 5 when the substrate was α,β-methylene-ATP. Thus, tritosomal membrane ATPase apparently exhibits a pH 8 optimum only when acting in concert with ADPase and AMPase in intact membranes. Rates of ATP hydrolysis to adenosine were also significantly greater in intact membranes than in Triton X-100-solubilized fraction. Centrifugation of Triton X-100-solubilized tritosomal membranes in sucrose density gradients showed that ATPase and ADPase activities sedimented to one peak, and that AMPase, acid phosphatase, and pyrophosphatase were grouped in another peak. Thus, tritosomal membrane ATPase activity was not due to the latter enzymes. The resulting purification was about fourfold for ATPase. The Mr for ATPase and ADPase was estimated to be about 65,000 and for AMPase, acid phosphatase, and pyrophosphatase about 200,000.     

Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon

Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.     

Differential stabilities of soil enzymes. Assay and properties of phosphatase and arylsulphatase.

Methods have been refined for the assay of phosphatase and arylsulphatase activities in soil, based on the chromogenic p-nitrophenyl ester substrates. Basic assay conditions have been defined, and pH optima and kinetic parameters have been determined. The enzymes follow Michaelis-Menten kinetics; this conclusion is based on three methods of analysis of data determined over a wide range of substrate concentrations. The enzyme activities are very stable to storage of wet soil for up to 4 weeks at soil temperatures and above. For example, phosphatase had a half-life of approximately 2 weeks at 50 degrees C; arylsulphatase was rather less stable. Both enzymes retained 80% of activity after incubation with pronase for 1 week at 25 degrees C. On the basis of this work and studies on other soil enzymes, it is concluded that remarkable stability is a general feature of soil enzymes.     

A HISTOCHEMICAL STUDY OF PHAGOCYTIC AND ENZYMATIC FUNCTIONS OF RABBIT MONONUCLEAR AND POLYMORPHONUCLEAR EXUDATE CELLS AND ALVEOLAR MACROPHAGES : I. Survey and Quantitation of Enzymes, and States of Cellular Activation

The cytochrome oxidase (CO), aminopeptidase (AMP), succinic dehydrogenase (SD), acid phosphatase, esterase, and alkaline phosphatase of rabbit mononuclear (MN) and polymorphonuclear (PMN) peritoneal exudate cells and pulmonary alveolar macrophages (AM) - air dried on Mylar strips - were characterized by histochemical techniques with respect to stability, activators, inhibitors, and pH optima. A granule count method was established for the quantitation of these enzymes. For the acid phosphatase of MN, in which the most precise results were obtained, time, pH, substrate, and inhibitor curves resembled those commonly obtained biochemically. Five of these enzymes were usually more active in AM than MN, whereas the sixth, alkaline phosphatase, was not present in either cell type. AM also tended to consume more oxygen than MN and to divide more frequently. Since the most active cells in the population would be first involved in the host's defense against microbial agents, a comparison was made of the 10 per cent of the AM and MN with the highest enzymatic activities. No differences were found in the granule counts that were not reflected by the means. However, within a given AM population, cells containing ingested dust particles seemed to have higher enzymatic activities than those without particles. MN had greater acid phosphatase and SD activities than PMN and consumed more oxygen, but the CO, AMP, and esterase activites of both types of cells were of similar magnitude. PMN showed high alkaline phosphatase activity; MN showed none. A survey of the histochemical literature indicates that a positive correlation between the enzymatic and phagocytic activities of both MN and PMN exists in vivo.