Enzyme最新文献

Exposure of V79 cells to hyperoxia (80% O2) for 30 h increased the level of thymidine kinase, a deoxynucleoside salvage enzyme, by approximately 3-fold as compared to cells exposed to room air, but did not cause any significant change in deoxycytidine kinase, the other known deoxynucleoside salvage enzyme. Exposure of cells to anoxia, on the other hand, produced only a slight reduction in thymidine kinase activity. Perturbation in cellular metabolism following exposure to hyperoxia was indicated by marked inhibition of cellular growth and the presence of cellular hypertrophy. Although growth was also inhibited by anoxia, the cell size distribution was minimally altered. The effect of hyperoxia on thymidine kinase suggests that (1) this enzyme may play a role in the modulation of cellular hypertrophy and function following exposure to hyperoxia, and (2) analysis of relative levels of thymidine kinase and deoxycytidine kinase activities may be of value in differentiating between cellular hypertrophy and hyperplasia under some circumstances.

The regulation of liver L-threonine deaminase by different effectors--bile acids, bile pigments and monocarbon molecules--was investigated. Total inhibition of the enzyme was observed with physiological concentrations of bile acids and biliverdin. Purely competitive inhibition of the holoenzyme by several monocarbon molecules was demonstrated; the mechanism was partially competitive for bicarbonate. Inhibition was more pronounced in the case of the dialyzed enzyme. From the higher Km values for pyridoxal-5'-phosphate (PLP), obtained in the presence of the inhibitors, the results are explained on the basis of interference in the association reaction: apoprotein + PLP----holoenzyme. The various effects determined by bicarbonate may play a specific role in vivo since they occur at physiological concentrations of this compound.

Protein synthesis is controlled by the phosphorylation of proteins comprising the translational apparatus. At least 12 initiation factor polypeptides, 3 elongation factors and a ribosomal protein are implicated. Stimulation of translation correlates with enhanced phosphorylation of eIF-4F, eIF-4B, eIF-2B, eIF-3 and ribosomal protein S6, whereas inhibition correlates with phosphorylation of eEF-2 and the alpha-subunit of eIF-2. Strong evidence for regulatory roles exists for eIF-2, eIF-4F and eEF-2, whereas changes in other factor activities due to phosphorylation remain to be demonstrated. Regulation of the specific activity of the translational apparatus by phosphorylation appears to be a general mechanism for the control of rates of global protein synthesis, and may also play a role in modulating the translation of specific mRNAs.

Early development in many animals is programmed by maternal mRNAs inherited by the fertilized egg. Many of these RNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation or fertilization. Polyadenylation plays a major role in controlling the translation of maternal mRNA during these times of development. Polyadenylation, in turn, is dependent upon two cis elements that reside in the 3'-terminal region of responsive mRNAs. In two cases, the factors that interact with these regions have been examined. The half-life of maternal mRNA also is regulated by polyadenylation, which again is controlled by 3'-terminal cis elements. The recent literature covering these topics is reviewed.

Succinylacetone (SA) is known to be a potent inhibitor of delta-aminolevulinic acid dehydratase (ALAD) in the liver. We have examined the effects of SA on the rat renal cortical enzyme, our observations indicating very different behaviour of renal versus hepatic ALAD with SA treatment. While the temperature response of ALAD in both tissues was similar, addition of 4 mmol/l SA inhibited liver ALAD at 37 and 55 degrees C and enhanced renal ALAD activity 2- to 3-fold at each temperature. This increase in renal ALAD was progressive with SA concentrations form 1 to 10 mmol/l. A pH titration curve for both liver and kidney ALAD showed the hepatic enzyme to have a single pH optimum, while the renal enzyme had two, each of which was distinct from that in liver. Kinetic studies with and without 4 mmol/l SA over a 50-fold ALA concentration range indicated SA-induced enhancement of renal ALAD over the entire range at both pH optima. Using 14C-labelled ALA, we have confirmed these observations made on the basis of a colorimetric assay for PBG, the enzyme product. We conclude that renal ALAD may be a different molecular species from the liver enzyme. Further studies may clarify the significance of these observations to renal heme synthesis.

Lymphoid cell lines from patients with infantile (type-2) and juvenile (type 3) Gaucher disease have been established by Epstein-Barr virus transformation and investigated and compared with the adult phenotype (type 1) with the view to enzymology. The enzymatic defect in glucosylceramide(GlcCer)-beta-glucosidase activity was more severe in type 2 and 3 than in type 1 cells. The mutant GlcCer-beta-glucosidase from our studied type 2 lymphoid cells was profoundly labile at pH 4.0 and 37 degrees C, whereas the residual GlcCer-beta-glucosidase from type 1 and type 3 were stable similar to the normal enzyme. In contrast to the distinct stability of the GlcCer-beta-glucosidases from the three phenotypes, the acid lability of the nonspecific membrane-bound beta-glucosidases from type 1, 2 and 3 were quite similar.

Urinary excretion of alanine aminopeptidase, alkaline phosphatase, gamma-glutamyltransferase and N-acetyl-beta-D-glucosaminidase was determined in gel-filtered samples of morning random urine specimens of 442 subjects of various ages (5 days to 58 years). Enzyme excretion related to urinary creatinine (enzyme/creatinine ratio; U/mmol creatinine) significantly decreased with increasing age. Sex-related differences of some enzyme excretions were found in age groups over 6 years. From these investigations, we calculated upper reference intervals (97.5 percentiles) for 5 age-dependent groups of children and adolescents and for one group of adults.

The rapid and dramatic induction of heat-shock proteins is accomplished by regulatory mechanisms acting at many different levels. Here we review current knowledge of two cytoplasmic mechanisms employed during the response in the fruit fly Drosophila melanogaster. (1) Heat-shock messages are translated with high efficiency during heat shock while most normal cellular messages are inactive. Sequences in the 5'-untranslated leader of heat shock mRNAs govern their preferential translation. (2) The messages for heat-shock proteins are unstable at normal temperatures. During heat shock, however, they are very stable and accumulate in large numbers. Sequences in their 3'-untranslated regions play a major role in determining their stability.

The pattern of protein synthesis changes soon after fertilization of clam oocytes. The most abundant of the mRNAs whose translation increases at this time encode ribonucleotide reductase and the A- and B-type cyclins. These mRNAs have been cloned and sequenced, yet their sequences do not show regions of similarity that could explain the masking mechanism. However, these mRNAs retain their 'masked' state in cell-free translation assays and their translation can be activated by gel filtration in high salt, which probably removes repressor proteins. A 'competitive unmasking' assay was used to identify the protein-binding regions of each mRNA. This involved adding short segments of antisense RNA that annealed to the mRNA and displaced the repressors. The unmasking regions in ribonucleotide reductase and cyclin A mRNAs revealed by this assay are 120-140 nt long and are located in the central portions of the 3' non-coding regions.