Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition of cytokinin (N6-delta 2-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa) were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate kinase (EC 2.7.2.3), and one was identified as the beta-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed. Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts.
Cotyledons of Cucurbita maxima Duch. seedlings were provided with (14)C-labeled amino acids for 12 h. Besides the bulk of labeled amino acids the sieve-tube exudate also carried labeled proteins. 80% of the incorporated radioactivity was found in the P-protein, 20% in a neutral protein, and traces were found in acidic proteins after fractionation on diethyl-aminoethyl cellulose columns. The radioactive elutes were characterized by autoradiographs of both disc- and sodium dodecyl sulfate-gelelectropherograms, and by isoelectric focusing. The P-protein fraction appeared with the void volume from the diethylaminoethyl-cellulose column. Obviously, this is the protein that gels when oxidized and that is reversibly precipitable giving rise to filaments when processed for electron microscopy. Its main component has a molecular weight of 115,000 Dalton. By isoelectric focusing this fraction separated into 3 proteins with isoelectric points of 9.8, 9.4, and 9.2. The isoelectric point 9.2-protein probably is identical with an oligomer of a 30,000 Dalton protein with neutral isoelectric point, which keeps 20% of the incorporated label. Microautoradiographs suggest that the labeled proteins were synthesized in companion cells. The results indicate that P-protein of Cucurbita maxima is synthesized continuously in mature phloem. It can be assumed that P-protein has a relatively high turn-over rate. Therefore it seems unlikely that P-protein is a "structural" protein.
The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.
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