Results of our conclusive study on urinary enzyme stability during sample storage are reported. We measured alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) in morning urines from 9 healthy normal subjects immediately after collection and throughout a 1-year storage at -70 and -20 degrees C. AAP proved to be quite stable at -70 degrees C (99.2% of the basal value at the end of the year). NAG is partially preserved (84.1% of the basal value) at -70 degrees C, but significantly decreased (50.4%) at -20 degrees C.

A sensitive and precise automated assay of urinary lactate dehydrogenase (EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1) and gamma-glutamyltransferase (EC 2.3.2.2) is described. For this purpose, we used a BM/Hitachi System 704 model and reagents for automated analysis of serum enzymes from Boehringer Mannheim. However, the schedules of enzyme chemistry parameters recorded by the autoanalyzer and the spectrophotometric calibration are reprogrammed to meet requirements deriving from urine adoption and to optimize the enzyme assay in this unusual medium.

Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl-CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.

An enzyme activity which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-Glu6,Ala3,Tyr1 (EAY; M(r) 47,000) has been demonstrated in the antral and body mucosa of the rat stomach. The distribution of this tyrosylprotein sulfotransferase was similar to that of the Golgi marker enzyme, glycoprotein sulfotransferase, and its activity from body mucosa was 23% higher than that from the antrum. The optimum for tyrosylprotein sulfotransferase activity was obtained at pH 6.8, in the presence of 0.5% Triton X-100, 20 mmol/l MnCl2, 50 mmol/l NaF, 2 mmol/l 5'-AMP, and 1 mmol/l DTT, whereas Ca2+, Mg2+, Cu2+, Zn2+, EDTA, NEM, NaCl and Na2SO4 were inhibitory. The apparent Km of the sulfotransferase for EAY was 1.5 x 10(-6) mol/l and for PAPS 0.75 x 10(-6) mol/l. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition as compared to that required for phenol sulfotransferase inhibition. The tyrosine sulfation by the tyrosylprotein sulfotransferase was independent of the sulfation of carbohydrate residues in mucous glycoproteins and glycolipids, thus indicating that the identified sulfotransferase is specific for sulfation of the tyrosyl residues in the peptide core.

Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.

We measured the cholinesterase activity in morning urines from 63 insulin-dependent diabetics and 27 controls. The total esterase (TotE) activity (Ellman's method) has been divided into aliesterase (AliE), pseudocholinesterase and acetylcholinesterase by means of two inhibitors, eserine and quinidine. Diabetics were divided in 2 groups according to the urinary albumin/creatinine ratio (mg/mmol, < 2 in group 1, > 2 in group 2). The urinary cholinesterase behavior was correlated with that of a known tubular lysosomal hydrolase, N-acetyl-beta-D-glucosaminidase (NAG). Compared to normals, in addition to a significant increase in urinary NAG in diabetes (in group 2 more than in group 1), TotE and AliE were also significantly raised (+36% and 109% of the controls, in group 1 as much as in group 2).

Rat kidney sialidase levels have been reported to be markedly altered in pathological states such as diabetes. This was associated with a modification of sialic acid levels. Therefore, it was interesting to study the variations of kidney sialidase and sialyltransferase activities and sialic acid content according to sex and age. This was carried out from birth to 210 days of age. The substrates used were sialyl alpha(2-3)[3H]-lactitol for sialidase activity, asialofetuin and [14C]-CMPNeu5Ac for sialyltransferase activity. In males sialidase activity increased until 32 days then slightly declined. In females, the activity increased and leveled off at 135 days of age. Higher sialidase activity was observed in females than in males from 56 days of age. Gonadectomy had no effect on this activity. In both sexes, sialyltransferase activity decreased markedly with age. This activity was higher in females than in males, whereas sialic acid levels varied only moderately with age and were slightly higher in females.

Endopeptidase 24.11 (EC 3.4.24.11) enzymatic activity was spectrofluorimetrically measured in human urine, using a synthetic peptidic substrate. Urinary endopeptidase 24.11 output (Uendo) was determined in 24-hour urine samples of 10 kidney transplant recipients during the first 2 weeks after surgery. In 9 patients, a large increase in Uendo levels was noted during the 1st and/or the 2nd postoperative days (mean +/- SEM of peak Uendo 624 +/- 122 micrograms/24 h, p = 0.0003 as compared to 239 +/- 20 micrograms/24 h in a healthy control population). This occurred whether patients received OKT3 (n = 6) or cyclosporine A (n = 3) as primary immunosuppression. Uendo returned to normal between the 3rd and the 5th postoperative day. We conclude that renal transplantation is associated with an early and marked release of endopeptidase 24.11 in urine. This could be due to the potentially toxic effects of ischemia and/or immunosuppressive drugs on the proximal tubular epithelium. The clinical usefulness of urinary endopeptidase 24.11 as a marker of tubular injury remains to be assessed.

A new method for the assay of maltase and sucrase is reported. The method makes use of mutarotase, hexokinase and glucose 6-phosphate dehydrogenase as ancillary enzymes. The reaction is linear at least up to a delta E/min of 0.13.

6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330-fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg2+ and NADPH and has a Michaelis-Menten constant of 10 microM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.

In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.

Acatalasemia was detected in 2 sisters of a Hungarian family. The pedigree of the family showed hypocatalasemia in the children of the patients and in 1 of their brothers, while the other members of the family had normal blood catalase activity. The biochemical characterization (catalase activity, electrophoretic migration, isoelectric point and enzyme stability) of the blood as well as tissue catalase of the acatalasemic patients yielded a catalase form which did not differ from normal.