Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

Constancy of rDNA Content during Dedifferentiation of Carrot and Jerusalem Artichoke Explants

When carrot explants were cultured with phytohormones, DNA synthesistook place synchronously in the explants and a satellite DNAwith a heavier density in CsCl than the bulk DNA replicatedin the earliest phase of the first replication period. The earlyreplicating carrot satellite consisted of a component havingan identical density to carrot rDNA and another component havinga density between the p-value of carrot rDNA and that of thebulk DNA. DNA-rRNA hybridization was used to explore the possibilitythat this early replication of the satellites leads to amplificationof rDNA in the explant cells, in which massive ribosome synthesisis known to occur. The results showed that there was neitheramplification nor underreplication of rRNA genes during callusformation and its growth. Experiments with explants of Jerusalem artichoke tuber, whichare well known as a synchronous replication system, showed thata component slightly heavier than the bulk DNA was synthesizedat the early phases of the first replication period. However,the density of this early replicating satellite differed fromthat of artichoke rDNA. DNA-rRNA hybridization experiments againshowed no gross changes of rDNA content during dedifferentiationof this plant system. (Received September 30, 1981; Accepted January 5, 1982)     

The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

Disulfide bonds of purothionine, a lethal toxin for yeasts.

Purothionin isolated from commercial wheat flour contained several components and two of them (A-I and A-II) were isolated in pure form by CM-52 column chromatography. Each component contained 45 amino acid residues with a 4 disulfide bonds. Purothionin A-II was digested with trypsin and thermolysin to isolate cystine peptides. These were separated and purified by chromatography on an SP-Sephadex column, and paper electrophoresis and chromatography. A peptide containing a -Cys-Cys- sequence was hydrolyzed with 10 N sulfuric acid. Amino acid compositions and partial sequence studies of the cystine peptides and their performic acid-oxidized peptides revealed the positions of all 4 disulfide bonds in purothionin A-II. They were formed between residues 3 and 39, 4 and 31, 12 and 29, and 16 and 25. The results of a partial study of purothionin A-I are also presented.     

The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Studies on algal cytochromes. III. Amino acid sequence of cytochrome c-553 from a brown alga, Petalonia fascia

The amino acid sequence of a photosynthetic cytochrome c-553 isolated from a brown alga, Petalonia fascia was determined by BrCN fragmentation and a solid phase Edman degradation. The cytochrome contains 85 amino acid residues, giving a molecular weight of 9,803. The complete amino acid sequence is as follows: Val-Asp-Ile-Asn-Asn-Gly-Glu-Ser-Val-Phe-Thr-Ala-Asn-Cys-Ser-Ala-Cys-His-Ala-Gly -Gly-Asn-Asn-Val-Ile-Met-Pro-Glu-Lys-Thr-Leu-Lys-Lys-Asp-Ala-Leu-Glu-Glu-Asn-Gl u-Met-Asn-Asn-Ile-Lys-Ser-Ile-Thr-Tyr-Gln-Val-Thr-Asn-Gly-Lys-Asn-Ala-Met-Pro-A la-Phe-Gly-Gly-Arg-Leu-Ser-Glu-Thr-Asp-Ile-Glu-Asp-Val-Ala-Asn-Phe-Val-Ile-Ser-Gln-Ser-Gln-Lys-Gly-Trp. The highest homology was found between the sequences of cytochromes c-553 of P. fascia and Alaria esculenta, the next between those of P. fascia and Porphyria tenera.     

Analyses of oligosaccharides by tagging the reducing end with a fluorescent compound. I. Application to glycoproteins.

The reducing end sugar of an oligosaccharide and 2-aminopyridine were linked by means of reductive amination with sodium cyanoborohydride. The fluorescent derivative of the oligosaccharide thus obtained, which had a positive charge, was subjected to two-dimensional paper electrophoresis. In the first direction, the sugar derivative moved according to its degree of polymerization, and in the second direction, it moved according to the structure of the borate complex. In this way fluorescent derivatives of saccharides were mapped on a sheet of paper. The method was applied to some known mono- and oligosaccharides and to the saccharides obtained by nitrous deamination of the oligosaccharide portions of glycoproteins (fetuin, Take-amylase A, and ovalbumin). The fingerprints thus obtained were characteristic of the chemical structures of the original oligosaccharides.