ABSTRACT
Synthesis of beta-maltosides, p-nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside, based on interaction of hepta-acetate-beta-D-maltosyl fluoride with the corresponding trimethylsilyl ethers of p-nitrophenol and 4-methylumbelliferone is described. 2-Chloro-4-nitrophenyl-beta-D-maltoside was synthesized by interaction of hepta-acetate-alpha-D-maltosyl bromide with 2-chloro-4-nitrophenol in two phase system using phase transfer catalyst. The method of assay of neutral alpha-glucosidase from human kidney and urine using synthesized beta-maltosides (p-nitrophenyl-beta-D-maltoside, 2-chloro-4-nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside) as substrates and beta-glucosidase as an auxiliary enzyme is proposed. The method is simple, convenient and 10-fold more sensitive than the commonly used alpha-glucosidase assay procedure with the corresponding synthetic alpha-glucosides, p-nitrophenyl-alpha-D-glucoside and 4-methylumbelliferyl-alpha-D-glucoside. A modification of the method, with p-nitrophenyl-beta-D-maltoside as substrate, was applied to the semi-automatic assay of urinary alpha-glucosidase in 96-well microtitre plates.
Subject(s)
Glucosides/chemical synthesis , Hymecromone/chemical synthesis , Nitrophenols/chemical synthesis , alpha-Glucosidases/metabolism , Glucosides/chemistry , Humans , Hymecromone/chemistry , Kidney/enzymology , Methods , Nitrophenols/chemistry , alpha-Glucosidases/urineABSTRACT
Use of antibodies towards neutral alpha-glucosidase from human kidney brush border enabled to estimate distinctly the activity of acid alpha-glucosidase in urine of healthy persons and patients although the activity of neutral enzyme in urine exceeded markedly the acid enzyme activity. Simultaneous use of antibodies to both these enzymes permitted to estimate separately the activity of acid and neutral alpha-glucosidases in a mixture. Accuracy of these estimations was confirmed after consecutive use of these antibodies.
Subject(s)
Clinical Enzyme Tests , Glucan 1,4-alpha-Glucosidase/urine , Glucosidases/urine , Immune Sera , alpha-Glucosidases/urine , Glomerulonephritis/diagnosis , Glucan 1,4-alpha-Glucosidase/immunology , Glycogen Storage Disease Type II/diagnosis , Humans , Precipitin Tests , alpha-Glucosidases/immunologyABSTRACT
As shown in studies of 123 patients with various nephropathies, activities of alpha-glucosidase and N-acetyl-beta-D-hexosaminidase in urine of the patients correlated with severity and stages of these diseases; at the same time, simultaneous estimation of both enzymatic activities in urine was shown to be more informative in diagnostic of kidney impairments. Low values of correlation coefficient (r = 0.35 +/- 0.09) between daily excretion of protein and the activity of neutral alpha-glucosidase in urine showed that the enzyme activity did not depend on proteinuria and was independent test for kidney impairment. Estimation of alpha-glucosidase activity could be used for control of the therapy; the enzymatic activity in urine correlated distinctly with the clinico-laboratory patterns of the patients studied.
Subject(s)
Clinical Enzyme Tests , Kidney Diseases/diagnosis , alpha-Glucosidases/urine , beta-N-Acetylhexosaminidases/urine , Acute Kidney Injury/diagnosis , Adolescent , Adult , Diagnosis, Differential , Female , Glomerulonephritis/diagnosis , Humans , Kidney Failure, Chronic/diagnosis , Male , Middle AgedABSTRACT
Distinct correlation was found between the activity of neutral alpha-glucosidase in urine and the degree of kidney impairment in 76 patients and in 15 healthy persons. In the patients with non-impaired kidney functions the activity of the enzyme in urine varied within the normal limits (from 12 to 39 microM/hr/mmole of creatinine; normal value being 14.6-15.9 microM), in the patients with moderately expressed disfunction of kidney--from 40 to 79 microM/hr/mmole of creatinine. The enzymatic activity, exceeding 80 microM/hr/mmole of creatinine (up to 227 mM), was observed in cases of severe impairment of the kidney functions. High activity of alpha-glucosidase, correlating with the severity of kidney impairment was found in urine of patients with pyelonephritis albeit the content of protein was quite normal. Interrelationship between the activity of alpha-glucosidase in urine and the state of kidney functions enables to conclude that the enzymatic activity depends on the degree of kidney impairment under various pathological conditions and that estimation of the enzyme activity in urine may be important for diagnosis of kidney diseases as well as for the control of treatment efficiency.
Subject(s)
Clinical Enzyme Tests , Glucosidases/urine , Kidney Diseases/diagnosis , alpha-Glucosidases/urine , Adolescent , Adult , Creatinine/urine , Female , Glomerulonephritis/diagnosis , Glomerulonephritis/enzymology , Humans , Hypertension, Renal/diagnosis , Hypertension, Renal/enzymology , Kidney Diseases/enzymology , Male , Middle Aged , Pyelonephritis/diagnosis , Pyelonephritis/enzymologyABSTRACT
In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.
Subject(s)
Glucosidases/isolation & purification , Kidney Cortex/enzymology , alpha-Glucosidases/isolation & purification , Cell Membrane/enzymology , Cytosol/enzymology , Drug Stability , Hot Temperature , Humans , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Microvilli/enzymology , Molecular Weight , Substrate Specificity , alpha-Glucosidases/metabolismABSTRACT
Multiple forms of neutral alpha-glucosidase were found in human urine. In a number of individuals three enzyme forms were detected, in other persons--four forms. The alpha-glucosidase I and 2 were present in urine as well as in kidney of some individuals, whereas in urine of other persons only alpha-glucosidase I was found. Both forms of neutral alpha-glucosidase I and 2 from human urine exhibited the maximal activity at pH 5.75 = 6.5, had a similar Km value (0.73 mM) with maltose as a substrate and did not differ in their properties from the corresponding forms of neutral alpha-glucosidase in a soluble fraction from human kidney.
Subject(s)
Glucosidases/genetics , Polymorphism, Genetic , alpha-Glucosidases/genetics , Humans , Isoenzymes/genetics , Isoenzymes/urine , Kinetics , Molecular Weight , alpha-Glucosidases/urineABSTRACT
It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.
Subject(s)
Chlamydiaceae , Fibroblasts/enzymology , Glycoside Hydrolases/metabolism , Acid Phosphatase/metabolism , Animals , Chick Embryo , Cytosol/enzymology , Female , Galactosidases/metabolism , Glucosidases/metabolism , Glucuronidase/metabolism , Lysosomes/enzymology , Mannosidases/metabolism , Vitelline MembraneABSTRACT
The data obtained show that most part of the activity of neutral alpha-glucosidases from human kidney is observed in the particle fraction, and only approximately 15%--in supernatant. Soluble neutral alpha-glucosidases have at least 4 different forms, as it is shown by means of their fractionation on Sephadex G-150, Bio-Gel P-200 and by polyacrylamide gel electrophoresis. Four forms are different in their molecular weight, electrophoretic mobility and substrate specificity. Two of the forms have molecular weight of 310000 and 110000. All the neutral alpha-glucosidases except high molecular weight form (greater than 400000) were retarded on column of Sephadex G-150.
