Canadian journal of biochemistry最新文献

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.

Rapidly sedimenting endoplasmic reticulum (RSER), which is known to be a complex between endoplasmic reticulum and mitochondria, was isolated from rat liver and purified through a sucrose density gradient by centrifugation according to a well established procedure previously published by G. C. Shore and J. R. Tata. This complex was characterized by microsomal (NADPH-cytochrome c reductase) and mitochondrial (succinate-cytochrome c reductase and NADP-isocitrate dehydrogenase) marker enzymes and was examined for the ability to synthesize microsomal lipids and mitochondrial polyglycerophosphatides. Results of these experiments showed that the RSER is capable of synthesizing key microsomal lipids, i.e., phosphatidic acid, phosphatidylcholine, and neutral lipids, as well as mitochondrial phosphatidylglycerosphate and phosphatidic acid, phosphatidylcholine, and neutral lipids, as well as mitochondrial phosphatidylglycerophosphate and phosphatidylglycerol. Furthermore, the level of synthesis of these lipids paralleled the level of activity of microsomal and mitochondrial marker enzymes found in the RSER preparation. Details of these experimental findings and some implications are discussed in view of the possible functional role of RSER.

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.

An enzymatic orthophosphate removal system is described which can be effectively used to continuously remove orthophosphate from biochemical samples. The phosphorolysis of nicotinamide riboside is catalyzed by calf spleen nucleoside phosphorylase to give ribose-1-PO4 and nicotinamide along with a proton. At pH 8 the production of ribose-1-PO4 from orthophosphate is essentially quantitative. This reaction can be monitored optically or by 31P nuclear magnetic resonance (NMR). Equations are given for determining the time required to remove a given amount of phosphate from a typical NMR sample with a known amount of nucleoside phosphorylase. The effects of a competing orthophosphate-producing reaction are considered.

The quaternary structures of ribulose-1,5-bisphosphate carboxylase-oxygenase from cold-hardened and unhardened Puma rye were examined by two-dimensional gel electrophoresis according to the method of O'Farrell. The results indicate that major changes in charge heterogeneity occur in the large subunit of this enzyme during growth at cold-hardening temperatures. The extent of charge heterogeneity decreased upon adaptation of Puma rye to cold-hardening temperatures. In addition to charge heterogeneity, molecular weight heterogeneity was also evident In the large subunit polypeptides of the enzyme from cold-hardened and unhardened Puma rye.

The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R +/- oxygen and the 500 R +/- oxygen (1 R = 2.58 x 10(-4) C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 microgram/ml. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 micrograms/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.

The light-dependent transhydrogenase system of Rhodopseudomonas spheroides which consists of a peripheral protein factor and a membrane-bound component contains essential sulfhydryl groups that are sensitive to p-hydroxymercuribenzoic acid (PMB). There are two types of sulfhydryl groups required for light-dependent (LD) transhydrogenation. One type is associated with the membrane-bound component and participates in or influences the binding of one of the substrates, NADH. A second type is associated with the peripheral protein factor and is not involved with the binding of the substrates. These sulfhydryl groups are masked or buried in the protein and are only exposed upon treatment of the peripheral protein factor with urea or trypsin. The peripheral protein factor may be an integral part of the transhydrogenase complex or may be required for the energization process. This factor appears to play an important role in the activation and control of LD-transhydrogenation.

Genomic libraries of macro- and micro-nuclear DNA of the ciliate protozoan Tetrahymena thermophila were constructed in the bacteriophage vector lambda gt WES lambda B. Screening of these libraries with a probe for the repeated hexamucleotide sequence C4A2 showed many phage from the micronuclear library but few if any macronuclear sequences having homology to this probe. This is consistent with C4A2-repeating elements being present predominantly if not exclusively at or near the termini of macronuclear DNA. Sequences flanking C4A2-repeating elements were isolated from a number of purified phage and were used as hybridization probes to restriction endonuclease digested macro- and micro-nuclear DNA. These experiments revealed a repeated sequence family as well as unique sequences present only in micronuclear DNA. The repeated sequence element appears to be dispersed throughout the genome. Phage-containing individual members of this micronucleus limited sequence family were purified from the micronuclear library. Some of these phage contained sequences which hybidized to macronuclear DNA. These fragments therefore contain a "transition" region between micronucleus-limited sequences and sequences present in both nuclei.

Low doses (30-84 ergs/mm2, 1 erg = 10(7) J) of ultraviolet radiation (UV) caused severe inhibition of the proliferation of human lymphocytes in vitro. Greatest inhibition was produced when resting cells were irradiated immediately prior to stimulation with concanavalin A (Con A); this was true whether activation was measured by the incorporation of labelled leucine, uridine, or thymidine. If UV was applied at 44 h after culture in presence of Con A, the incorporation of [3H]thymidine measured 4 h later was seen to be inhibited but transcription and translation were scarcely affected. UV, applied after the appearance of the Con A induced thymidine transport system, did not inhibit its function. The disaggregation of chromatin and increase in nuclear volume characteristic of, and essential to, the proliferative response of lymphocytes were completely inhibited if the cells were irradiated before mitogen was added to the cultures, but were unaffected if irradiation occurred after 16 h of culture in presence of Con A. Cells irradiated with 84 ergs/mm2 at the onset of culture with mitogen did not show the early increase of cation pump function which is a characteristic of stimulated lymphocytes, when this was measured by means of 86Rb uptake after 2-4 h culture. The mitogen-stimulated activation of cation pump function has previously been shown to be unaffected by concentrations of cycloheximide and actinomycin D which produce virtually complete inhibition of protein and RNA synthesis, respectively. The major inhibitory effect of UV treatment of lymphocytes at onset of culture with Con A is therefore not on DNA or on DNA synthesis, but on some component(s) of the early activation process, possibly at the cell periphery; inactivation of this component prevents cells from proceeding into later stages of the proliferative pathway.