Enzyme最新文献

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.

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.

It is now well established that hepatocytes are the main liver cells responsible for the synthesis of plasma proteins produced by the liver. That these cells are not specialized in the production of the different plasma proteins is also well established. Presently the point still debated is whether a functional hepatocellular heterogeneity exists for plasma protein synthesis as for many other hepatocyte functions. Several physiological and pathological situations suggest that this heterogeneity takes place in the hepatocytes of two opposite hepatic lobular zones, the periportal and centrilobular zones. However, this zonal difference, which supposes different regulatory mechanisms, must be confirmed by techniques other than the now classical immunocytochemistry or the in situ hybridization technique recently proposed for the demonstration of mRNAs in hepatocytes. Another hepatocellular heterogeneity, the intercellular heterogeneity, which can be observed in the same lobular zone, is more difficult to analyze, but shows that from hepatocyte to hepatocyte a variation exists in the synthesis of a given plasma protein.

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.

The major cytosolic aldehyde dehydrogenase isozyme (ALDH1) exhibits strong activity for oxidation of retinal to retinoic acid, while the major mitochondrial ALDH2 and the stomach cytosolic ALDH3 have no such activity. The Km of ALDH1 for retinal is about 0.06 mumol/l at pH 7.5, and the catalytic efficiency (Vmax/Km) for retinal is about 600 times higher than that for acetaldehyde. Thus, ALDH1 can efficiently produce retinoic acid from retinal in tissues with low retinal concentrations (< 0.01 mumol/l). The gene for ALDH1 has hormone response elements. These findings suggest that the major physiological substrate of human ALDH1 is retinal, and that its primary biological role is generation of retinoic acid resulting in modulation of cell differentiation including hormone-mediated development.

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.

We analyzed the expression profile of isoenzymatic fractions of creatine phosphokinase (EC 2.7.3.2) isotypes MM, MB and BB in three cell lines derived from embryonic rhabdomyosarcomas and a normal counterpart cell line. Electrophoretic data showed that the BB fraction was consistently expressed de novo, in contrast with its counterpart in normal tissue. The BB fraction may serve as new tumoral marker for the diagnosis of rhabdomyosarcoma. In addition, the appearance of macrocreatine kinase type-1 in this type of neoplasm may serve to reinforce the diagnosis when rhabdomyosarcoma is suspected.