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C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.  相似文献
2.
Isolated intact chloroplasts from wall-less mutants of Chlamydomonas reinhardtii accumulate inorganic carbon (Ci) from the medium provided the cells had been adapted to low CO2 photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO2 or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast Ci accumulation from low CO2 grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for Ci accumulation by Chlamydomonas, it is proposed that CO2 diffuses into the cell and Ci accumulation occurs in the chloroplast.  相似文献
3.
A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.  相似文献
4.
A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO2 concentrations but can grow on elevated CO2. In a test cross, the high CO2-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO2 accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO2 by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO2 levels to an environment low in CO2. It is concluded that this mutant fails to induce the CO2 concentrating system and is incapable of adapting to low CO2 conditions.  相似文献
5.
Structural elucidation of an intensely blue fluorescent compound (A) formed from sepiapterin by Bacillus subtilis is described. The structure of the catabolite (A) was found to be 2-amino-6-(1-carboxyethoxy)-4(3H)-pteridinone (9) from both spectroscopic and degradation studies. This was confirmed by an unambiguous synthesis of 9. The stereochemical structure of the side chain at the 6-position of A was confirmed to be the L(or S) configuration, as in sepiapterin, by analysis of the lactic acid formed from A on acid hydrolysis. This suggests that the side chain is rearranged intact during the catabolic conversion of sepiapterin. A possible mechanism for the conversion is discussed.  相似文献
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