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The D1 protein of photosystem II is synthesized in precursor with a carboxyl-terminal extension. Interestingly, there is quite a range in chain length of the extension, which roughly depends upon the class of organisms. In cyanobacteria, e.g. in Synechocystis sp. PCC6803, the extension consists of 16 amino acid residues, seven residues longer than its counterpart in higher plants. In this study, we examined the D1 processing in Synechocystis sp. PCC6803 by pulse-chase experiments and detected a proteolytic intermediate of this process. This finding suggests that the elongated extension in this organism is excised with a sequential two-step proteolysis, which differs markedly from the manner observed in higher plants.

Solvent extraction at approximately 4 degrees C in complete darkness, and subsequent analysis by high-pressure liquid chromatography (at approximately 4 degrees C) using an apparatus equipped with a two-dimensional diode-array detector, spectroscopically identified 15-cis-beta-carotene in the reaction center (RC) of spinach photosystem II (PS II). The result revises a previous conclusion that beta-carotene bound to the PS II RC takes the all-trans configuration [Fujiwara, H., Hayashi, H., Tasumi, M., Kanaji, M., Koyama, Y. and Satoh, K. (1987) Chem. Lett. 2005-2008], and generalizes the concept established for purple photosynthetic bacteria that 15-cis-carotenoid is naturally selected by the photosynthetic reaction centers.

A cross-linked product between the D1 and D2 subunits was generated by treating isolated spinach PS II reaction center with a bifunctional cross-linker, 1,6-hexamethylene diisocyanate. To obtain information on the contact site(s) between D1 and D2 proteins, the cross-linked product was cleaved by CNBr, the resultant fragments were separated using a reverse-phase HPLC, and then the partial sequence of the cross-linked polypeptide fragments were determined. By comparing the results with the deduced amino acid sequence of the D1 and D2 proteins from spinach, it is concluded that the C-terminal domains of the D1 subunit (D308-A334) and that of the D2 subunit (Y297-L353) are in close proximity.

Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same Km and Vmax values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the Vmax value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e., binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.

Two distinct cross-linked products containing psbI gene product were generated in the photosystem II reaction center complex from spinach by treatment with bifunctional reagents directed to amino groups. The first product, which was generated by a 3,3'-dithiobis(succinimidyl propionate) treatment, is deduced to be formed between the epsilon-amino group of Lys3 of the psbI gene product, and a side-chain amino group present on the stromal extension of the D2 protein. The CNBr cleavage analysis of the cross-linked product predicted that the amino group of the D2 protein engaged in the cross-linking is either one of the three lysine residues on the N-terminal fragment (from N-terminus to Met19) or the Lys268 on the 6th fragment (from Val248 to Met275). The second product, which was also generated on the stromal side by a 1,6-hexamethylene diisocyanate treatment preferentially in alkaline conditions, is predicted to be formed between the epsilon-amino group of Lys3 of the psbI gene product and the N-terminal alpha-amino group of the alpha-subunit of cytochrome b559.

Phosphoinositide metabolism elicited by anti-CD9 monoclonal antibody, a well-characterized platelet activator, was studied using acetylsalicylic acid-treated human platelets. TP82, which is an anti-CD9 monoclonal antibody, induced classical phosphatidylinositol 4,5-bisphosphate hydrolysis, as monitored by intracellular Ca2+ mobilization and phosphatidic acid production, and synthesis of phosphatidylinositol 3,4-bisphosphate, which is a major component of newly-described 3-phosphorylated inositol phospholipids produced during platelet activation. These changes were severely inhibited by 1 microM staurosporine, a potent, though non-selective, protein kinase inhibitor, which also abolished TP82 induction of tyrosine phosphorylation of multiple platelet proteins. Protein-tyrosine phosphorylation appears necessary to initiate both the classical phosphoinositide turnover and synthesis of the newly-described 3-phosphorylated inositol phospholipids in anti-CD9 monoclonal antibody-induced platelet activation.

In an in vitro translation experiment using spinach chloroplasts, a novel protein band of about 17.5 kDa appeared by light to dark transition. The protein never accumulated in detectable amounts either in continuous illumination or in continuous darkness. The 17.5 kDa protein accumulated upon light-dark transition, on the other hand, disappeared by the subsequent illumination. Accumulation of the protein in light, however, was observed when stromal level of ATP in chloroplasts was lowered after preillumination by the addition of various chemical compounds which, irrespective of the mode of action, eventually decrease the ATP level, e.g. atrazine, carbonyl-cyanide-m-chlorophenyl hydrazone and glycerate. The dark-accumulated protein was concluded to be a translation intermediate of D1 protein based on the facts that this component precipitates with specific antibodies and is resistant to lysylendopeptidase treatment. The suppression by chloramphenicol of both appearance upon light-dark transition and disappearance by the subsequent illumination of the protein also supported this conclusion. The phenomenon was discussed in terms of pausing in the translation of psbA mRNA.

Chloramphenicol inhibited significantly but incompletely photosynthesis in leaf segments of rice. Fluorescence and polarographic experiments indicated that chloramphenicol competes with the CO2 reducing cycle for electrons from photosystem I because it serves as an electron acceptor of photosystem I and its reduction intermediate transfers its electron to molecular oxygen.

A pigment-protein complex consisting of D1 and D2 proteins, but depleted in the two lower molecular mass components of photosystem II, i.e. cytochrome b-559 and psbI gene product, has been isolated by octyl-beta-D-glucopyranoside treatment of the purified photosystem II reaction center complex from spinach [(1987) Proc. Natl. Acad. Sci. USA 84, 109-112], followed by separation by high performance liquid chromatography using a gel-permeation column (TSK G3000 SW). The isolated complex is photochemically active in the photoreduction of intrinsic pheophytin a under steady-state illumination, in the presence of dithionite and methyl viologen, and exhibits pigment stoichiometries similar to those in the untreated reaction center, indicating that the D1-D2 complex provides the site of primary photochemistry in photosystem II, as well as the principal binding sites of pigments in the reaction center.

Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.

Nucleoside-diphosphate (NDP)-kinase can be considered to be induced by human-type interferons (HuIFNs) rapidly, since an enzyme increase was detected within 2 h of incubation of HeLa S3 cells with HuIFNs, while incubation with heterologous mouse IFNs had no such effect. The enzyme increase induced by HuIFNs reached a plateau at 6 h after treatment. Actinomycin D (0.5 microgram/ml) significantly blocked the enzyme increase induced by HuIFNs in the cells. A possible biological role of the enzyme in the IFN-induced biochemical events is discussed.