Aggregation properties of beta-galactosidase of human urine and degradation of its natural substrates by a purified preparation of the enzyme.

Acid beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was purified to near homogeneity from normal human urine by two affinity chromatography steps. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the major protein band had an apparent molecular weight of 59000, thus being 5000 daltons smaller than the protein purified from human liver. Upon gel filtration on Sephadex G-150 column the purified enzyme had an apparent molecular weight of 70000 of pH 7.0. At pH 4.0 partial aggregation to a dimer of an apparent molecular weight of 150000 was found. Addition of 0.1 M galactose caused at pH 3.5, but not at pH 4.0 and 7.0, an increased formation of multimeric beta-galactosidase which eluted with the void volume of the column. Crude beta-galactosidase from human urine showed a higher aggregation tendency than the purified enzyme. None of the conditions produced an enzyme species of an apparent molecular weight of less than 40000. pH-activity profiles were measured against p-nitrophenyl-beta-D-galactoside, 3H-labelled GM1-ganglioside, [3H]keratan sulfate and the pentasaccharide O-beta-(1 leads to 4)-[6-3H]galactopyranosyl-O-beta-(1 leads to 2)-2-deoxy-2-acetamidoglycopyranosyl-O-alpha-(1 leads to 6)-mannopyranosyl-O-beta-(1 leads to 4)-mannopyranosyl-2-deoxy-2-acetamidoglucopyranoside. While p-nitrophenyl-beta-D-galactopyranoside and GM1-ganglioside were optimally hydrolyzed at pH 4.0, keratan sulfate and the pentasaccharide were optimally degraded at pH 4.3 and pH 5.0, respectively. With the chromogenic substrate and with GM1-ganglioside Km values of 0.33 mM were calculated. At pH 3.5 the hydrolysis of the synthetic substrate did not follow Michaelis-Menten kinetics. Two enzyme species appeared with Km values of 0.006 mM and 3.2 mM, respectively. The affinity of beta-galactosidase for [3H]keratan sulfate and the 3H-labelled pentasaccharide was at least one order of magnitude lower than for the amphiphilic substrates. Keratan sulfate and GM1-ganglioside did not act as competitive inhibitors of p-nitrophenyl-beta-galactosidase at the concentration tested. These findings could be explained by the existence of different binding sites for the substrates used.

Altered lysosomal glycohydrolase activities in juvenile diabetes mellitus.

Studies have been carried out on activities of lysosomal beta-N-acetylhexosaminidase (hex), beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu), and acid phosphatase (AP) in serum and urine from patients with juvenile diabetes and matched controls. There is a large increase in blood and urinary hex activity (the former presenting three distinct patterns of abnormality), a moderate increase in urinary beta-gal, and a small increase in urinary alpha-glu activity, but no elevation of blood or urinary AP in the diabetics. Urinary alpha-glu activity in the diabetics shows striking inhibition by glucose, and this may reflect a similar phenomenon in vivo. Although glycohydrolase activities are elevated in patients with no detectable microangiopathy, more striking changes may be observed in patients with severe small-vessel disease. These alterations may be associated with increased glycoprotein catabolism in the diabetic, an area in need of further studies in the human and experimental diabetic animal.

Characterization of the acid beta-D-galactosidases from human urine.

Acid beta-D-galactosidases (EC 3.2.1.23) from human urine samples have been characterized using GM1-ganglioside, asialofetuin, and 4-MU-beta-D galactopyranoside. Sepharose 6-B column chromatography of crude urine supernatant fluids resolved three forms of acid beta-D-galactosidase activity with apparent molecular weights of 500 X 10(3)--700 X 10(3) (I), 90 X 10(3)--120 X 10(3) (II), and 20 X 10(3)--27 X 10(3) (III), which hydrolyzed 4-MU-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin. The crude urine supernatant fluids and the separated forms of acid beta-D-galactosidase exhibited similar apparent KM values for the respective substrates. Starch gel electrophoresis of urine samples at pH 7.0 revealed a slow anodally migrating form of acid beta-D-galactosidase which electrophoretically corresponded to form I and a faster anodally migrating form corresponding to form II. Form III migrated as a composite of forms I and II suggesting that aggregation to the larger molecular weight activity forms occurred during starch gel electrophoresis. This report represents the first characterization of urinary acid beta-D-galactosidase with respect to naturally occurring glycolipid and glycoprotein substrates. In addition, data is presented to indicate that the enzyme may be composed of an enzymatically active form with an apparent molecular weight of 20 X 10(3)--27 X10(3), which is also capable of hydrolyzing the glycolipid and glycoprotein substrates.

Rapid colorimetric assay of beta-galactosidase and N-acetyl-beta-glucosaminidase in human urine.

Colorimetric methods using 4-nitrophenyl-glycoside substrates for the assay of beta-galactosidase and N-acetyl-beta-glucosaminidase in human urine are described. Interfering substances were removed by gel-filtration of urine. An unidentified low-molecular-weight inhibitor of N-acetyl-beta-glucosaminidase was found. Increased sensitivity of the methods was achieved by high sample volumes, small volumes of buffer to terminate the enzyme reaction and optimal substrate concentration and buffer pH. Incubation periods were shortened to 15 min. Both methods were designed as single-tube tests in which buffer-substrate solutions are prepared in bulk and aliquots are stored in individual containers at -25 degrees C. Under these conditions reagents were stable for at least six months. Precision of both methods was satisfactory. Estimates of normal limits are reported.

Urinary lysosomal enzyme excretion after renal allotransplantation.

Three urinary lysosomal enzymes, beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal) and N-acetyl-beta-D-glucosaminidase (NAG), were measured in twenty-one renal allograft recipients to evaluate their role in the diagnosis and prediction of rejection episodes, and in the prediction of eventual graft outcome. A fluorometric assay using methylumbelliferone substrates was used to measure the three enzymes in morning urine samples and enzyme activity was defined in terms of urine creatinine concentration. Urinary NAG levels increased significantly in 13/16 first rejection episodes and 4/4 instances of acute tubular necrosis and graft infarction. In 5 of the 16 first rejection episodes the NAG was predictive of the rejection. NAG was not useful in diagnosing second or subsequent rejections and beta-Gluc and beta-Gal were of little value in assessing any component of renal transplant pathology. As a prognostic index of eventual graft outcome, the peak urinary NAG was particularly encouraging. It correlated strongly with deterioration in graft function as time passed such that only 2/10 patients with peak NAG greater than 1400 Units had normal serum creatinines at 6 months post transplantation. Conversely 4/4 patients with peak NAG levels less than 700 Units had normal serum creatinine at that time. In our series the measurement of urinary NAG was a useful adjunct to the diagnosis of first rejections but appears to be more valuable in predicting graft outcome.

Diurnal variations of urinary enzyme excretion.

Variations in the urinary excretion of arylsulphatase A, beta-galactosidase, alpha-glucosidase and beta-glucuronidase throughout a 24-h period were studied in 8 healthy subjects. Urine was collected at 3-h intervals and enzyme activities were assayed after gelfiltration of the urine specimens. Significant intra-individual changes of the excretion of all 4 enzymes during the 24-h period were found. Enzyme output was high between 3 a.m. and 9 a.m. and low during the afternoon and evening hours. The most striking pattern was seen for arylsulphatase A. Diurnal variations of urinary enzyme excretion seemed not to be flow dependent. Both modes of expression of enzyme output (mU/min or U/g creatinine) gave corresponding results. It is concluded that for the measurement of the excretion of these enzymes urine should be collected during a fixed time interval, e.g. from 6 a.m. to 9 a.m.

Colorimetric assays for N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase in human urine using newly-developed omega-nitrostyryl substrates.

(1) The synthesis of 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (MNP-GlcNAc) and 2-methoxy-4-(2'nitrovinyl)-phenyl beta-D-galactopyranoside (MNP-Gal) as substrates for the assay of NAG and beta-d-galactosidase are described. (2) beta-Glycosidase activities were determined in random urine samples from normal males and females aged between 12 and 87 years and patients with renal disease. (3) Both the MNP N-acetylglucosaminide and MNP galactoside were stable indefinitely, if stored in the solid state at 4 degree C in the dark. (4) The effect of urinary inhibitors was minimized by diluting the urine in the assay procedure. A simple assay procedure has been developed using MNP substrates. A good correlation was found with established assays using 4-methylumbelliferyl and p-nitrophenyl glycosides. (5) The assay was readily automated and a good correlation was found between the automated and manual methods. (6) The assay of urinary glycosidase activity with MNP substrates is simple to perform and has been used successfully in the clinical chemistry laboratory.

Properties of the residual alpha-galactosidase activity in the tissues of a Fabry hemizygote.

The properties of the residual alpha-galactosidase activity in kidney, liver, spleen, fibroblasts and urine of a Fabry hemizygote have been studied using p-nitrophenyl-alpha-galactoside and 4-methylumbelliferyl-alpha-galactoside as substrates. In addition, alpha-galactosidase activity in urine has been determined with ceramidetrihexoside as substrate. The residual alpha-galactosidase activity of Fabry, measured with artificial substrate, is stimulated (6-35%) by myo-inositol and only slightly inhibited by melibiose (7-17%) in all the materials used. In contrast, the alpha-galactosidase of normal tissues and urine is inhibited (36-48%) by myo-inositol and inhibited to a much greater extent (40-50%) by melibiose. The KM for artificial substrate of the residual activity of Fabry is higher than that of the alpha-galactosidase in normal kidney, liver, spleen, fibroblasts and urine. The residual activity of Fabry is generally more stable to heating than the activity in the normal materials, although exceptions were noted. When these properties are compared with those of the alpha-galactosidase isoenzymes in normal tissues and body fluids, the residual activity of Fabry material seems to be very similar to the minor component of normal tissue (alpha-galactosidase B). Moreover, the pH optimum curve of this minor component and of the Fabry alpha-galactosidase in urine are similar, whereas the major isoenzyme (alpha-galactosidase A) shows a curve much more like that of normal urine. The findings with ceramidetrihexoside as substrate indicate a possible discrepancy. Alpha-Galactosidase A hydrolyses ceramidetrihexoside, Fabry urine preparation does not. However, alpha-galactosidase B of normal urine shows a slight but definite ceramidetrihexosidase activity. No contamination of the B preparation with alpha-galactosidase A could be detected. The minimum hypothesis, supported by most of the experimental evidence, is that the residual activity of Fabry and normal alpha-galactosidase B are identical.

The diagnostic value of urinary enzyme measurements in hypertension.

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.

Urinary lysosomal enzymes for detection of acute renal allograft rejection.

Six patients who received renal transplants were closely monitored to compare the sensitivity of urine levels of beta-galactosidase and N-acetyl-beta-glucosaminidase with conventional clinical and laboratory parameters in the detection of impending rejection. A rapid (60 minute), simple, accurate fluorometric assay was used to measure activities of both enzymes. Eighty per cent of ten rejection episodes were accompanied by a two- to sixfold increase in enzyme release. Parallel changes in serum creatinine levels and urinary volume occurred in six rejection episodes, but in two episodes, elevated urinary enzyme levels were observed two and four days prior to clinical evidence of rejection. It is concluded that urinary lysosomal enzyme measurements by fluorometric assay are valuable indicators of acute renal rejection, particularly when the diagnosis is not clearly established by conventional criteria that show only minimal changes. Continuing studies in a large group of renal transplant recipients are under way to evaluate the validity of this conclusion and to determine whether enzyme measurements, will, indeed, be indicative of early rejection.

Urinary enzyme excretion during renal papillary necrosis induced in rats with ethyleneimine.

N-Acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucosidase, acid and alkaline phosphatase were monitored in urine kidney homogenates and serum of rats with papillary damage induced with ethyleneimine. Serum urea levels, total protein in the urine and urine volume were monitored throughout the study. Histological studies showed that the injection of ethyleneimine caused immediate papillary necrosis, followed later by secondary cortical involvement. Minor papillary necrosis induced by a low dose (0.5 mul/kg) of ethyleneimine was characterised by a rise in urinary N-acetyl-beta-glucosaminidase activity which was followed later by an increase in the activity of the other enzymes monitored. More severe papillary necrosis induced with a higher dose of ethyleneimine (5.0 mul/kg) resulted in an immediate rise in the activities of all the urinary enzymes which then decreased only to rise again when cortical involvement occurred. Serum urea was unaltered but urine volume and protein were increased coincidentally with the urinary enzyme activities. The value of the assay of urinary enzymes in distinguishing papillary from glomerular and tubular damage is assessed. The possible relevance of the ethyleneimine model to the etiology of papillary nephropathy is discussed.

[Normal values of the urinary activity of 4 enzymes]. / I valori normali dell'attività urinaria di 4 enzimi.

The urinary activity of 4 enzymes (NAG, GLU, GAL, GRS) was investigated in 105 healthy subjects in order to evaluate the variability of standard levels and establish the degree of such variations in relation to sex, age, weight and height. The results obtained demonstrate that these enzymurias do vary in relation to the parameters examined. Age and sex produced the most pronounced variations though height and body weight also appeared to have some influence. The study of variations in standard levels is of value in the interpretation of pathological enzymurias.

Some urinary enzymes in bilharziasis.

Urinary alkaline phosphatase (ALP), acid phosphatase (ACP), aryl sulphatase (Ar. sulph.), beta-glucuronidase (beta-gluc.) and galactosidase were assayed in a group of Bilharzia haematobium patients and another group of healthy subjects (control group). The results for most of the determined enzymes revealed high activities as compared to the controls. The activity of acid phosphatase in male urine samples increased also, though not significantly. These elevated enzyme activities could be used to establish the diagnosis of schistosomiasis in patients whose urine contains no ova or when it is difficult to detect them. The results are discussed in the light of localization of each enzyme in the urinary tract as well as in other organs like the liver.