High-pressure liquid chromatography was used to separate chlorophyll derivatives in acetone extracts from senescing Citrus fruit peel, autumnal Melia azedarach L. leaves, and dark-held detached parsley (Petroselinum sativum L.) leaves. Chlorophyllide a and another polar, dephytylated derivative accumulated in large amounts in senescing Citrus peel, particularly in fruit treated with ethylene. Ethylene also induced a 4-fold increase in the specific activity of Citrus chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14). Detailed kinetics based on a hexane/acetone solvent partition system showed that the in vivo increase in dephytylated derivatives coincided with the decrease in total chlorophyll. Polar, dephytylated derivatives accumulated also in senescing Melia leaves. Senescing parsley leaves revealed a very different picture. The gradual disappearance of chlorophyll a was accompanied by an increase in pheophytin a and by the transient appearance of several phytylated derivatives. Only pheophytin a and an adjacent peak were left when all the chlorophyll a had disappeared. The pathways for breakdown of chlorophyll in the Citrus and parsley senescence systems are discussed.
A 71-kDa protein (P71) with properties similar to those ofthe Escherichia coli heat shock protein DnaK has been found in extracts of HeLa cells. P71 was copurified by ATP-agarose affinity chromatography with three additional proteins of the Hsp7O family. Of these proteins, only P71 crossreacted strongly with antiserum raised against purified DnaK, and both DnaK and P71 could be phosphorylated in vitro with [y-32]ATP in a reaction that was markedly stimulated by Ca2+. In HeLa cells, P71 was found to be concentrated in mitochondria. A protein similar to P71 was also found in calf liver and yeast mitochondria.A universal response of living organisms to a variety of stress agents including elevated temperatures is the synthesis of a specific subset of proteins (reviewed in ref. 1). The heat shock protein referred to as Hsp7O has been intensively studied because of its early induction and high rate of synthesis during stress. In eukaryotic cells, Hsp7O belongs to a large gene family with complex regulatory mechanisms (2-4). These proteins share a high degree of homology, and in addition, all organisms contain constitutively expressed members, suggesting that the proteins of this group share a similar role in normally growing and stressed cells (5). A current view is that one of the major functions of the Hsp7O family of proteins is to facilitate the proper folding of proteins, as may be required for their translocation across the endoplasmic reticulum and mitochondrial membranes (6-9). Proteins that are members of the Hsp7O family are found in diverse species including bacteria. In Escherichia coli, the product of the dnaK gene is a 72-kDa heat shock protein homologous with the Hsp7O family of eukaryotes (1). Previous studies on DnaK showed that it is a phosphoprotein and that purified preparations of DnaK could be autophosphorylated in vitro (10, 11). The present report shows that one of the Hsp7O-related proteins, a 71-kDa protein (P71), is found in the mitochondria of eukaryotic cells and shares some of the characteristics of DnaK. During the preparation of this manuscript, Craig et al. (12) reported the sequence of the yeast SSCI gene. This gene codes for a Hsp7O protein that appears to be similar to P71, since it is localized in mitochondria and has a high degree of homology to DnaK. MATERIALS AND METHODSHeLa (S3) cells were grown in suspension at 37°C to a density of 6-8 x 105 cells per ml in Joklik's modified minimal essential medium (13) supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were harvested by centrifugation at 200 x g for 5 min and were washed in 10 mM Hepes-KOH (pH 7.5) containing 0.15 M NaCl. Cell pellets were either used immediately for cell fractionation or frozen in liquid nitrogen for protein purification.For cell fractionation, cells (1 x 109) were suspended in 15 ml of sucrose buffer (0.25 M sucrose/10 mM Hepes-KOH, pH 7.5), the suspension was forced through a 26-gauge needle twice, and the lysate was centrifuged at 1000 x g for 10 min at 4TC. The pellet was resusp...
The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with [35S~sulfate at both the normal growth temperature (210C) and an elevated temperature (36TC). Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature. Three of the proteins appear to be immunologically related to the ubiquitous -70-kDa heat-shock protein (Hsp7O) family. One protein of 68 kDa was found in the cytoplasm (P68Cyt) and was the major heat-shock protein in Euglena gracilis. Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68,,) and chloroplasts (P7ch), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK. Like DnaK, P68mit and P70ChI could be phosphorylated in vitro with [v-32PIATP in a reaction that was stimulated by Ca2. A protein with characteristics similar to those of P70Chi was also found in chloroplasts isolated from maize and spinach.
Isogenic strains of Synechococcus PCC 7942 were genetically engineered so that copy I of the gene psbA was mutated at specific sites. These mutations resulted in replacements of Ser 264 by Gly or Ala and of Phe 255 by Tyr or Leu in the D1 protein. The mutants were resistant to herbicides inhibiting electron transfer in photosystem II. All mutants exhibited alterations in the stability of QB- as demonstrated by a temperature downshift, to various extents, of the in vivo thermoluminescence emission. Measurements of the light-dependent turnover of D1 showed a marked decrease in the t 1/2 of this protein in the mutants as compared to wild-type, under low to medium light intensities. A correlation was found between the degree of pertur bation in the QB- stability and the rate of acceleration in the turnover of D1. These data pro vide a direct evidence for the overlapping binding sites for the plastoquinone B and herbicides in the D1 protein. In addition these data indicate a close link between QB- destabilization in reaction center II and the mechanism controlling the light-dependent turnover of D1. Based on these results and previous work we suggest that destabilization of the semireduced quinone, facilitates a light-induced damage in D1 which triggers its degradation.
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