Enzyme In Urine Research Articles

N-acetyl-beta-D-glucosaminidase assay in urine: urea inhibition.

Urea inhibition of urinary N-acetyl-beta-D-glucosaminidase (NAG) was examined with human isoenzymes and total enzyme in urine. The fluorometric substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide (MUNAG) and the colorimetric substrate m-cresolsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide (MCPNAG) were used to determine substrate kinetics and apparent urea inhibition kinetics. Apparent KmS of 0.47 (MUNAG) and 0.35 mmol/L (MCPNAG) for human NAG isoenzyme A and 0.46 (MUNAG) and 0.30 mmol/L (MCPNAG) for human NAG isoenzyme B were determined. Apparent Kis of approximately 100 mmol/L for urea inhibition of both isoenzymes in either substrate and apparent Kis of 79 (MUNAG) and 91 mmol/L (MCPNAG) for NAG in dilute urine samples were found. The potential for urea inhibition at physiological concentrations of urea and at normal assay dilutions therefore exists. Inhibition at these dilutions was minimal when substrate concentrations of 2 x Km or greater were used, but the substrate MUNAG at 1/2 x Km gave the highest sensitivity and lower blanks and allowed better quantitation of low activity samples. Within-run CVs of 3.2% with MUNAG and 10% with MCPNAG were found for low activity samples.

Distribution of 5’ -nucleotidase in the tissues of sheep and the effect of kidney and liver lesions on the activity of the enzyme in plasma and urine

The distribution of 5'-nucleotidase (5'-NT) activity in the tissues of the sheep differs from that of gamma glutamyl transferase (GGT). Nevertheless, both enzymes are released into the plasma of sheep which have been infected with Fasciola hepatica or in which the bile duct has been ligated. In a sheep dosed with sporidesmin, a fungal toxin which damages the biliary tract, there was an increase in GGT activity in the plasma and urine but no change in 5'-NT activity. Neither enzyme was released into the plasma or urine of sheep dosed with carbon tetrachloride. In sheep with renal tubular necrosis and hepatocellular necrosis caused by dosage with hexachlorobutadiene, both enzymes were released into the plasma, and GGT, but not 5'-NT activity was found in urine. In sheep with tubular necrosis of the kidney caused by the administration of mercuric chloride, GGT activity, but not 5'-NT, increased in the plasma and urine.

Biochemical properties and pathophysiological significance of angiotensin converting enzyme in human urine

Biochemical properties and pathophysiological significance of angiotensin converting enzyme in human urine

Studies on human urinary arginine esterases

A new arginine ester hydrolyzing enzyme in the human urine (human urinary arginine esterase, HUAE) was found. The content levels of this new enzyme, determined by N-α-tosyl-L-arginine methyl ester (Tos-Arg-Me) hydrolyzing activity, were measured to be 0.34 to 0.86 nmol/min/ml of human urine and the percentage of content level of this enzyme was calculated to be about 10 to 20 per cent of the total Tos-Arg-Me hydrolyzing activity. HUAE was partially purified and then separated into two forms (HUAE-1 and -2) by DEAE-cellulose chromatography. The approximate molecular weights of HUAE-1 and -2 were estimated to be 4.8×104 and 3.0×104 by Sephadex G-100 gel filtration, respectively.

Clinical study on the angiotensin I-converting enzyme in human urine. (I) Partial purification, enzymic characteristics and excretion in normal subjects

The angiotensin I-converting enzyme in normal human urine was partially purified with ammonium surfate (3.2M), DEAE-Cellulose ion exchange chromatography (0-0.5M NaCl gradient), and Sephadex G 200 gel filtration. The enzyme was separated into three forms which had different molecular weights of 700000, 290000 and 40000, respectively. The enzymic biochemical characteristics of these three enzymes, however, were identical with regard to their inhibitory effects (bradykinin potentiator c, arg-pro-pro, o-phenanthroline and EDTA), Cl- dependency, optimal pH (8.3) and temperature (37 degrees C), and Km value (2.3mM). The enzymic activity was determined in five normal subjects in three conditions of dietary sodium intake (51, 153 and 340mEq for 5 days, respectively). The enzymic activity correlated well with the concentration of the excreted sodium (r = 0.87, p less than 0.001). There was no significant relation between the enzymic excretion and the concentration of the excreted potassium, nor between the activity and the creatinine excretion. It is suggested that the origin of urinary angiotensin I-converting enzyme is the kidney, and that the enzyme might regulate sodium excretion in cooperation with renal kallikrein-kinin system.

Variants of 7-glutamyl transferase in human urine

When dialyzed human urine was subjected to ultracentrifugation, gamma glutamyl transferase activity amounting to one-third or less of the total activity was found associated with the microsomal fraction and the remaining in the 100 000 X g supernatant. By ion-exchange chromatography on DEAE-cellulose at pH 7.5, the urinary enzyme was resolved into two fractions. The less anionic form (eluted with 0.15 mol/l NaCl) corresponded to the 100 000 X g supernatant fraction. The more anionic form (eluted with 0.5 mol/l NaCl) corresponded to the microsomal fraction. On chromatography on concanavalin A-sepharose, the urinary transferase activity separated into the unbound fraction and the bound fraction (eluted with α-methyl- d-glucoside). The bound fraction was retained more tightly on DEAE-cellulose (eluted with 0.5 mol/l NaCl). The unbound fraction had weaker interaction with the ionic-exchanger (eluted with 0.15 mol/l NaCl). Each of two fractions separated by affinity chromatography on concanavalin A-sepharose, resolved into two bands on cellulose acetate electrophoresis at pH 7.5. In addition, during gel chromatography on Sephadex G-200, they separated into a high molecular weight (>200000) fraction and a low molecular weight (55000) fraction. The band that stayed at the origin during electrophoresis was high molecular weight in nature, whereas the fraction that moved towards the anode was found to be the smaller form of the enzyme. The study of the variants of urinary gamma glutamyl transferase was not found to be of clinical significance. The nature and origin of the variant forms of the enzyme in urine are discussed.

Ureteral contractions induced by rat urine in vitro: probable involvement of renal kallikrein.

Rat urine, even at a 1:10 final dilution in Tyrode's solution, stimulates contraction of the ureteral musculature in vitro. This effect can be ascribed to the presence of kallikrein or a kallikrein-like enzyme in urine. Isometric contractions of ureters were prevented by previous addition of aprotinin to the organ bath. Urine also lost its activity after inactivation of enzymes by heat or acid treatment.

PHARMACOLOGICAL STUDIES ON EXPERIMENTAL NEPHRITIC RATS (9). CHANGES IN ACTIVITIES OF URINARY ENZYMES IN THE MODIFIED TYPE OF MASUGI’S NEPHRITIS AND THEIR SOURCES

Using a modified model of Masugi’s nephritis of rats, various enzymatic activities in urine, serum and renal tissue (glomeruli or cortex) were determined at appropriate intervals after the administration of anti-kidney serum and compared with the urinary protein content and the kidney weight. In the urine, alkaline phosphatase (Al-Phosase), acid phosphatase (Ac-Phosase) and N-acetyl-β-glucosaminidase (NA-β-Gase) activities remarkably increased after the induction of nephritis, reached their peaks on the 10th day and reverted to almost the normal levels on the 30th day. The patterns of time course of these enzymatic activities were similar to patterns seen in the urinary protein content and the kidney weight. In the serum, the Al-Phosase activity decreased slightly, while NA-β-Gase activity increased slightly. The Ac-Phosase activity in serum remained at normal levels during the experimental periods. In the glomeruli, the bound activities of these three enzymes decreased with nephritis, showing a negative correlation with results in the urine. On the other hand, fibrinolytic activities in the urine (plasmin-like enzyme) and renal cortex (plasminogen activator) also paralleled the urinary protein content and the kidney weight in the course of the disease. These results suggest that the Al-Phosase, Ac-Phosase and NA-β-Gase excreted into urine in cases of nephritis may be mostly derived from damaged renal cells and one part of Al-Phosase may also come from the plasma. Moreover, the increase of plasmin-like enzyme in urine is considered to be due to the increase of plasminogen activator in the renal cortex. Thus, the determination of these enzymatic activities in the urine should be useful for evaluating effects of drugs for the treatment of nephritis.

Vergleichende Untersuchung zur diagnostischen Wertigkeit von Diskelektrophorese der Urinproteine und N-Acetylglucosaminidaseausscheidung zur Erkennung von tubulären Nierenschädigungen bei chronischer Polyarthritis

A method is described for the determination of the enzyme N-acetyl-beta-D-glucosaminidase in gel-filtered urine. The individual reaction parameters were tested, and the reliability of the method was determined. The stability of the enzyme in urine was investigated at different temperatures over a 6 week period. The excretion of N-acetyl-beta-D-glucosaminidase, like the analysis of urinary proteins by disc electrophoresis, is a sensitive parameter for renal damage. The two methods were compared, using 50 random urine samples from patients with chronic polyarthritis; both methods were found to have equal diagnostic value.

Pharmacological studies on experimental nephritic rats (9). Changes in activities of urinary enzymes in the modified type of Masugi's nephritis and their sources.

Using a modified model of Masugi's nephritis of rats, various enzymatic activities in urine, serum and renal tissue (glomeruli or cortex) were determined at appropriate intervals after the administration of anti-kidney serum and compared with the urinary protein content and the kidney weight. In the urine, alkaline phosphatase (Al-Phosase), acid phosphatase (Ac-Phosase) and N-acetyl-beta-glucosaminidase (NA-beta-Gase) activities remarkably increased after the induction of nephritis, reached their peaks on the 10th day and reverted to almost the normal levels on the 30th day. The patterns of time course of these enzymatic activities were similar to patterns seen in the urinary protein content and the kidney weight. In the serum, the Al-Phosase activity decreased slightly, while NA-beta-Gase activity increased slightly. The Ac-Phosase activity in serum remained at normal levels during the experimental periods. In the glomeruli, the bound activities of these three enzymes decreased with nephritis, showing a negative correlation with results in the urine. On the other hand, fibrinolytic activities in the urine (plasmin-like enzyme) and renal cortex (plasminogen activator) also paralleled the urinary protein content and the kidney weight in the course of the disease. These results suggest that the Al-Phosase, Ac-Phosase and NA-beta-Gase excreted into urine in cases of nephritis may be mostly derived from damaged renal cells and one part of Al-Phosase may also come from the plasma. Moreover, the increase of plasmin-like enzyme in urine is considered to be due to the increase of plasminogen activator in the renal cortex. Thus, the determination of these enzymatic activities in the urine should be useful for evaluating effects of drugs for the treatment of nephritis.

DETECTION AND ACTIVITY OF BLOOD GROUP B-GENE ASSOCIATED α-GALACTOSYLTRANSFERASE IN HUMAN URINE

1. α-galactosyltransferase which participates in the biosynthesis of blood group B substance was found in urine from group B and AB healthy persons of both secretors and non-secretors.2. The activity of α-galactosyltransferase in urine of healthy variant Bm person was lower than that found in normal group B person. The level of this enzyme in urine of A1Bm persons must be much lower than that in mormal AB. The enzyme activity was not detected by the method of group O to group B transformation of erythrocytes.

Angiotensin I-converting enzyme in human urine

It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 ± 0.04 (S.E.M.) units/day ( n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium ( r = 0.76, p < 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (>400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl −3 dependency, pH optimum and K m value.

Studies on human urinary trypsin inhibitor 1. Its modification on treatment of urine with acid

A urinary trypsin inhibitor with an estimated molecular weight of 67,000 (UTI-1) was isolated from normal human urine by ammonium sulfate precipitation and gel filtration on Sephadex G-100. When urine was acidified prior to ammonium sulfate precipitation, two new inhibitors (UTI-11 and UTI-111) with molecular weights of 45,000 and 23,000 were obtained. The modification of UTI-1 seemed to be due to a heat-labile enzyme in native urine, since it could be prevented by heating the urine at 100 °C for 15 min. The heat labile enzyme might be a protease which was active under acidic condition, such as uropepsin.

A direct assay for urinary kallikrein.

The first protein-binding assay is described for the direct determination of kallikrein in rat urine. Isolation of urinary kallikrein and preparation of its specific antibody have been previously described. Outlined here are the methods for 3H-labelling of kallikrein, for isolation of 3H-labelled immunoreactive enzyme, and for separation of free and antibody-bound kallikrein with the aid of double antibody. Assay and equilibrium conditions were characterized and a protocol is presented for the measurement of kallikrein concentration. The direct assay is sensitive, accurate and it correlates well (r = 0.81) with a functional assay. The assay may hold promise to determine catalytically active and inactive enzyme in urine, tissues and biological fluids.

Crystallization of an α-Amylase and Evidence for the Presence of a Glucose-forming Enzyme in Human Urine

Crystallization of an α-Amylase and Evidence for the Presence of a Glucose-forming Enzyme in Human Urine

Bestimmung der l-alanyl-peptidhydrolase (Alanin-aminopeptidase, aminosäure-arylamidase) im menschlichen harn

A method formerly described for the determination of l-alanyl-peptide hydrolase (AAP) in serum has been developed for the estimation of this enzyme in urine. l-Alanine-(3-naphthylamide serves as the substrate. The preparation of urine, the basis of reference, the optimal collection period and collection time are investigated. In healthy persons the excretion of l-alanylpeptide hydrolase is subject to circadian variations. Furthermore, it depends on age. In the group of 18–30 years there is a sex difference.

Relationship Between Blood Amylase and Urinary Amylase in Man

Somogyi1 has shown that the amylase content in the blood is rather constant for the individual, while the variations from individual to individual are considerable. The study of the amylase content of the urine, however, reveals great irregularity in the same individual at various periods of the day, without any apparent regularity in the variations. The analytical methods used were those described by Somogyi.1,2,3 We found it very important to take into consideration the optimum pH (6.8-7.4) and the optimum salt concentration (0.25-0.4% NaCl).In healthy human beings the concentration of the enzyme in the urine is greater than in the blood, the ratio of urine amylase to blood amylase being usually between 2:1 and 6:1. These fluctuations in ratio occur in the same person, often in the course of a single day. Great as these variations are, we find that on the whole there is a parallelism between the blood and urinary amylase concentrations; i. e., high blood amylase usually goes with a high urinary amylase,...