ABSTRACT
The type A or 'acid' and type B or 'neutral' beta-galactosidase activities have been measured in post-mortem liver samples from individuals dying of non-genetic diseases and patients dying of ganglioside storage disease other than GM1 gangliosidosis. The type A activities fell within the established normal range in all samples. The type B activities showed a biomodal distribution suggesting the occurrence of two distinct populations of human individuals. The greater proportion had activities within the range 11.67 pkat/mg of protein (+/- 3.33, S.D.), while others had lower activities in the range 0.48 pkat/mg of protein (+/- 0.38, S.D.). No clinical symptoms were associated with the much lower type B beta-galactosidase activities and it appears that this beta-galactosidase deficiency could be found in the original tissues. Methods of screening for type B beta-galactosidase deficiency are described and the significance of this enzyme deficiency is discussed.
Subject(s)
Isoenzymes/isolation & purification , Lactose Intolerance , Liver/enzymology , Chromatography, DEAE-Cellulose , Electrophoresis, Starch Gel , Gangliosidoses/enzymology , Glycoside Hydrolases/analysis , Hot Temperature , Humans , Hydrogen-Ion Concentration , NeuraminidaseABSTRACT
1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.
Subject(s)
Galactosidases/isolation & purification , Liver/enzymology , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Starch Gel , Humans , Hymecromone , Isoelectric Focusing , Isoenzymes/isolation & purification , Neuraminidase/pharmacologySubject(s)
Galactosidases/metabolism , Isoenzymes/metabolism , Liver/enzymology , Animals , Chlorides/pharmacology , Cross Reactions , Drug Stability , Enzyme Activation , Galactosidases/antagonists & inhibitors , Hot Temperature , Humans , Hydrogen-Ion Concentration , Molecular Weight , Rabbits/immunologyABSTRACT
Hexosaminidase C was separated from human brain supernatant by immunoadsorption of the A and B forms on to a column of immobilized antibody followed by preparative starch-block electrophoresis. There were some differences in the properties of hexosaminidase C preparations after each of these stages, shown by comparison of their heat-inactivation characteristics and filtration through Bio-Gel P-200. The C form prepared by both separation steps had properties which differed markedly from those of the A and B isoenzymes; its molecular weight was much larger, greater than 200000, it had optimum activity between pH6 and 7 and could not be successfully eluted from DEAE-cellulose, even with high salt concentrations, or from Sephadex G-200. These results seem to support the proposal that the C form is under a separate genetic control from the others.
Subject(s)
Brain/enzymology , Hexosaminidases/isolation & purification , Adsorption , Animals , Antibodies , Chromatography, DEAE-Cellulose , Electrophoresis, Starch Gel , Filtration , Hot Temperature , Humans , Hydrogen-Ion Concentration , Isoenzymes/isolation & purification , Molecular Weight , Rabbits/immunologySubject(s)
Brain/enzymology , Carbohydrate Metabolism, Inborn Errors/enzymology , Hexosaminidases/metabolism , Lipidoses/enzymology , Animals , Cellulose , Chromatography, DEAE-Cellulose , Chromatography, Ion Exchange , Electrophoresis , Electrophoresis, Starch Gel , Female , Humans , Hydrogen-Ion Concentration , Isoenzymes/metabolism , Kinetics , Liver/enzymology , Organ Specificity , Pedigree , Placenta/enzymology , Polysaccharides , Precipitin Tests , Pregnancy , Rabbits/immunology , Spectrometry, FluorescenceSubject(s)
Glycoside Hydrolases/blood , Child , Chromatography, DEAE-Cellulose , Diabetes Mellitus/enzymology , Electrophoresis, Starch Gel , Female , Hexosaminidases/blood , Hexosaminidases/isolation & purification , Hot Temperature , Humans , Kinetics , Lipidoses/enzymology , Male , Pregnancy , Protein DenaturationSubject(s)
Glycoside Hydrolases/urine , Kidney Diseases/enzymology , Animals , Arsenicals , Galactosidases/urine , Glucosidases/urine , Glucuronidase/urine , Hexosaminidases/urine , Immune Sera , Isoenzymes/urine , Kidney Diseases/chemically induced , Kidney Diseases/urine , Kidney Tubules/pathology , Male , Rabbits , RatsSubject(s)
Galactosidases/urine , Glycoside Hydrolases/urine , Kidney/surgery , Postoperative Complications/urine , Adult , Circadian Rhythm , Electrophoresis , Fluorometry , Gels , Glucosamine , Humans , NephrectomySubject(s)
Galactosidases/urine , Glycoside Hydrolases/urine , Kidney Diseases/enzymology , Electrophoresis , Glomerulonephritis/urine , Glucosamine , Glucosidases/urine , Humans , Hypertension, Renal/enzymology , Inflammation/enzymology , Kidney/anatomy & histology , Kidney Failure, Chronic/enzymology , Nephrotic Syndrome/urine , Organ Size , Pyelonephritis/enzymology , Urinary Bladder Neoplasms/enzymology , Urinary Calculi/enzymologySubject(s)
Galactosidases/metabolism , Glucosidases/metabolism , Glycoside Hydrolases/metabolism , Kidney/enzymology , Adult , Cell Nucleus/enzymology , Chromatography, Ion Exchange , Electrophoresis , Female , Fluorometry , Galactosidases/urine , Glucosidases/urine , Glycoside Hydrolases/isolation & purification , Glycoside Hydrolases/urine , Humans , Hydrogen-Ion Concentration , Kidney/cytology , Kidney Diseases/diagnosis , Kinetics , Lysosomes/enzymology , Male , Microsomes/enzymology , Mitochondria/enzymology , Protein Denaturation , Spleen/enzymology , Time FactorsABSTRACT
1. Free and total activities of beta-glucosidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and beta-glucuronidase have been determined fluorimetrically in five subcellular fractions of rat kidney. 2. The beta-glucosidase activity appeared in the soluble fraction, beta-glucuronidase had the distribution pattern of a lysosomal enzyme, and both beta-galactosidase and N-acetyl-beta-glucosaminidase had bimodal distributions. 3. Two types of beta-galactosidase activity were found: a sedimentable type, having optimum pH3.7, mol.wt. about 80000 and slow electrophoretic mobility at pH7.0 in starch gel; and a soluble type of much faster mobility, having optimum pH5.5-6.5 and mol.wt. about 40000. 4. Evidence is presented that the beta-glucosidase and the soluble type of beta-galactosidase are the same enzyme. 5. Most of the N-acetyl-beta-glucosaminidase activity was in the lysosome-rich fractions, but a significant proportion occurred in the microsomal fraction in a non-latent form. 6. The use of beta-galactosidase and N-acetyl-beta-glucosaminidase as lysosomal marker enzymes is complicated by the possible presence of multiple forms, but this limitation does not apply to beta-glucuronidase in the rat kidney.
Subject(s)
Galactosidases/analysis , Glucosidases/analysis , Glucuronidase/analysis , Glycoside Hydrolases/analysis , Kidney/enzymology , Animals , Cell Nucleus/enzymology , Chromatography, Gel , Electrophoresis , Kidney/cytology , Lysosomes/enzymology , Male , Microsomes/enzymology , Mitochondria/enzymology , Rats , UltracentrifugationABSTRACT
1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl, beta-glucosaminidase were estimated in normal and pathological rat urine, with 4-methylumbelliferyl glycosides as substrates. 2. Kidney damage induced by injections of uranium nitrate, mercuric chloride, potassium dichromate or 4-nitrophenylarsonic acid causes a marked increase in the urinary excretion of all four enzymes. 3. The rise in beta-glucosidase activity was associated with the appearance of a new urinary enzyme species, which was examined by starch-gel electrophoresis, DEAE-cellulose chromatography and filtration on Sephadex G-75 and G-200. 4. This enzyme appears to be identical with its counterpart in the kidney, and it is suggested that it arises in the urine as a result of renal tubular breakdown. 5. The other glycosidases examined also show some physical similarities to the corresponding enzymes of the rat kidney.
Subject(s)
Galactosidases/urine , Glucosidases/urine , Glucuronidase/urine , Glycoside Hydrolases/urine , Kidney Diseases/metabolism , Kidney/enzymology , Animals , Chromatography , Chromatography, Gel , Electrophoresis , Hydrogen-Ion Concentration , Kidney Diseases/chemically induced , Kinetics , Male , RatsABSTRACT
1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7.0 indicated slow- and fast-moving components of rat-kidney beta-galactosidase. 3. The fast-moving component is also associated with the total beta-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the beta-glucosidase component is a small acidic molecule, of molecular weight approx. 40000-50000, with optimum pH5.5-6.0 for beta-galactosidase and beta-glucosidase activities. 5. The major beta-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3.7.