Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.
A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.
ABSIRACIChloroplast envelope membranes isolated from Pisum satium seedlings have been foud to cotain a Mg-ATPase activity (specifi activity 50-175 nanomoles per minute per milligram protein). The ATPase had a broad pH optimum between 7.0 and 9.5. The activity was not inhibited by oligomycin, N,N'-dicyclohexylcarbodlimide, ouabain, or antibodies directed agpinst chloroplast coupling factor 1; nor was the activity stimulated by monovalent cations. However, the ATPase was inhibited by vanadate, molybdate, and adenylyl imidodiphosphate.The ATPase hydrolyzed a broad range of nucleoside triphosphates, but did not hydrolyze ADP, AMP, or pyrophosphate. The K,. for Mg-ATP was determined to be 0.2 millimolar. The ATPase was found to be distinct from ADPase and pyrophosphatase activities also present in pea envelope membranes.The ATPase was determined to be located on the inner membrane of the envelope after resolution of inner and outer membranes by sucrose density gradient centrifugation.
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