Enzyme & protein最新文献

Carbamoylphosphate synthetase I (CPS I), a urea cycle enzyme, is located almost exclusively in the mitochondria of hepatocytes. The enzyme is unique in that it constitutes about 2-6% of total liver protein and is composed of a large subunit of 160 kD. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for measurement of the enzyme in plasma using an antibody against the rat enzyme. In galactosamine-induced rat acute hepatitis, plasma concentration of CPS I that was 1-2 micrograms/ml blood before the treatment, increased up to 125 micrograms/ml blood in 24 h after the treatment and decreased to a near control level in 72 h. Plasma concentration of ornithine carbamoyl-transferase (OCT), another urea cycle enzyme, reached a maximum in 24 h and then decreased a little more rapidly than that of CPS I. On the other hand, alanine aminotransferase activity reached a maximum in 36 h and decreased to a normal level in 96 h. In immunoblot analysis, the native CPS I polypeptide of 160 kD and its fragments of 140 and 125 kD were detected 24-48 h after the treatment. When purified rat CPS I and bovine OCT were injected intravenously into rats, the enzymes disappeared from blood roughly exponentially with apparent half-lives of about 67 and 18 min, respectively. Development of an ELISA for human CPS I and determination of the serum enzyme in various liver diseases remain to be performed.

In A-431 cells, stimulation of P2-purinergic receptors with extracellular ATP caused production of inositol 1,4,5-trisphosphate (InsP3), followed by mobilization of Ca2+ from intracellular stores; Ca2+ influx from the extracellular fluid and breakdown of phosphatidylcholine (PtdCho) also accompanied this InsP3/Ca2+ signalling. When A-431 cells were pretreated with pertussis toxin (PTX), production of InsP3 and elevation of cytosolic Ca2+ were strongly inhibited. PTX also inhibited the Ca2+ influx, but the effect was much weaker than that for InsP3/Ca2+ elevation. No inhibitory effect was observed in ATP-stimulated PtdCho breakdown. These results suggest that there is a system(s) which mediates the functions of P2-purinergic receptors in addition to PTX-sensitive G-proteins.

To investigate proximal tubular dysfunction under hyperglycemic status, we infused 10% glucose solution into male Wistar rats with and without 0.16% phloridzin (a specific inhibitor of proximal tubular glucose transportation) and measured the urinary excretion rates of enzymes that are derived predominantly from proximal tubules. We used 10% mannitol solution and 0.9% saline as controls. Urinary excretion levels of N-acetyl-ss-D-glucosaminidase, alanine aminopeptidase, gamma-glutamyl transpeptidase and dipeptidyl aminopeptidase IV were significantly increased in the 10% glucose-loaded group. In contrast, these increased enzyme excretions were not observed in the 10% mannitol or 0.9% saline-loaded group. Moreover, addition of 0.16% phloridzin to 10% glucose solution completely prevented these increases in N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase and gamma-glutamyl transpeptidase excretion, and slightly decreased dipeptidyl aminopeptidase IV excretion. At the end of the infusion study, a rise in renal cortical sorbitol concentration of the 10% glucose-loaded group was about 2 times higher than the 0.9% saline or 10% mannitol-loaded group. However, in the group that received both glucose and phloridzin, elevation of renal cortical sorbitol concentration was not observed. This study showed that glucose-load results in both abnormal enzymuria and renal cortical sorbitol accumulation; they were completely prevented by phloridzin.

Liver cirrhosis in man is often associated with hyperinsulinemia but its pathogenesis is still unexplained. To investigate whether insulin degradation is impaired in cirrhotic liver, the specific insulin-degrading enzyme activity (EC 3.4.22.11) was assayed in liver cytosol of rats with CCl4-induced liver cirrhosis. No difference was found between liver cytosol of cirrhotic and control rats. The results show that experimental CCl4-induced liver cirrhosis does not damage the specific insulin-degrading activity and support the hypothesis that impaired hepatic insulin handling is not an important cause of hyperinsulinemia in liver cirrhosis.

The present study was designed to investigate the effects of grape seed tannins on rat intestinal alkaline phosphatase (AP), sucrase and dipeptidyl peptidase IV (DPP IV) activities. An experiment was performed in vivo by dietary supplementation with 2% tannins; this diet was tested on an experimental group of rats; a control group received a diet without tannins. After 31 days, tannins intake significantly decreased middle-jejunal AP from 123 to 45 mU/mg protein and sucrase activities from 310 to 195 mU/mg protein, while no significant difference appeared at the duodenal stage (p < 0.05). Ileal DPP IV activity was also significantly reduced (p < 0.05) from 190 to 110 mU/mg protein after tannin intake. Using in vitro experiments on purified brush border membranes, AP activity was found to be inhibited by grape tannins; this inhibition was prevented by the detergent Triton X-100. The addition of pancreatic-biliary (PB) juice to the incubation medium prevented or reversed the tannin-inhibited enzyme activity. The present data indicate that in the duodenal lumen, alkalinity and detergency from the PB secretion neutralized the ability of tannins to inactivate brush border hydrolase activities and suggest that enzyme inhibition took place once bile salts were reabsorbed while moving down the gut. This was confirmed by in vitro experiments where sucrase and DPP IV activities inhibited by grape seed tannins were largely recovered after the addition of PB juice to the incubation medium.

Ornithine carbamoyltransferase (OCT), a urea cycle enzyme, is located almost exclusively in liver mitochondria. We designed a sensitive enzyme-linked immunosorbent assay (ELISA) of serum OCT protein using an antibody against purified bovine enzyme. OCT protein measured using this method showed a good correlation with OCT activity (r = 0.961), and was much higher in patients with liver diseases than in the controls. Measurements of serum OCT protein in 442 healthy blood donors gave the upper limit of normal range of 23 ng Eq/ml (equivalent to the bovine enzyme) for males, 8 ng Eq/ml for females, and 16 ng Eq/ml for males plus females. The values differ significantly between the sexes and depending on ages. This ELISA system is expected to be used as a pertinent liver function test.

A unique protein has been detected that is associated with the differentiation of diffuse large cell lymphoma (DLCL). The WSU-DLCL human cell line was cultured in the absence or presence of the biological agent, Bryostatin 1 (Bryo1). Cellular proteins of parent and differentiated WSU-DLCL cells were analyzed using one- and two-dimensional polyacrylamide gel electrophoresis (1D and 2D PAGE). In the 1D PAGE, a unique protein band of molecular mass approximately 47 kD was detected in the differentiated, but not the parent cells. Amino acid sequence of the band indicated the presence of more than one protein. The 2D PAGE analysis showed that one of the proteins of interest had an isoelectric point of 7.4. Partial amino acid sequencing of the spot by tryptic digest showed 100% homology with alpha-enolase. alpha-Enolase is a nonneuronal enzyme involved in the glycolytic pathway. This is the first report on the induction of alpha-enolase in human DLCL after treatment with the natural biological agent, Bryo1. We suggest that alpha-enolase may play a significant role in the differentiation of lymphoma in man.

We used near and far UV spectrophotometry for the re-evaluation of the molar extinction coefficient at 280 nm (epsilon 280) of pulmonary angiotensin-converting enzyme (ACE), for the determination of its tryptophan and tyrosine contents and to follow-up guanidine denaturation. ACE purity was assessed by both SDS-PAGE and capillary electrophoresis performed in denaturing conditions. The maxima of the near UV spectrum of purified ACE, dissolved in phosphate buffer pH 6.5, was at 279 nm; with an estimated M(r) of 160 kD, epsilon 280 of native ACE was 1.5 +/- 0.05 x 10(5) (mol/l)-1 x cm-1. Denaturation of ACE by 6 mol/l guanidine hydrochloride produced a hypochromic effect of 23% at 280 nm and led to a blue shift of 3.5 nm. In guanidine solution, absorbance measurements at 288 and 280 nm predicted a ratio of 1 between tyrosine and tryptophan, whereas it was 1.8 with the measure of the amplitude of the spectral bands at 283 and 292 nm of the second derivative of the near UV spectrum. Unfolding of the peptide chain in 6 mol/l guanidine was also well characterized by the second derivative of the far UV spectrum, in parallel with the complete loss of enzymatic activity although the protein remained whole as judged on SDS-PAGE. We also re-evaluated ACE zinc content by atomic absorption spectroscopy and demonstrated that ACE molecule obviously contains two zinc atoms.

Freezing of serum samples at -30 degrees C without protective agents is the simplest and least expensive method of storage in serum banks. We investigated the stability of creatine kinase (CK) in human sera after freezer storage under such conditions for 24 h (n = 30) or for 2 or 4 weeks (n = 99). CK activity was measured in fresh sera and compared to matched thawed sera after freezer storage at the designated time intervals. The enzyme's median activity decreased significantly after 24 h, 2 weeks, and 4 weeks of freezer storage by 2.6, 5.9, and 8.3%, respectively (p < 0.0001, r = 0.99). Sex or high CK initial values had no significant effect on these results. We conclude that freezer storage of serum at -30 degrees C, even for short periods, causes a steady and significant decline in CK activity. These results should be taken into consideration when analyzing CK activity in frozen sera for research or clinical purposes.

The ocular lens grows by laying down new cells on top of old in a differentiation process that results in loss of protein-synthesizing capacity, but preservation of the cells themselves for the lifetime of the organism. The transparency and refractive index of the lens depend on protein integrity and longevity, yet proteolysis is needed for normal growth and development. Therefore, control of proteolysis must be stringent. Here we review the structural features and major proteolytic enzymes of the lens and the properties of the bovine lens multicatalytic proteinase complex, including native and SDS-PAGE patterns, and activation and inhibition by cations, amphiphilic molecules and temperature.