No abstract

Hepatitis B surface proteins play a central role in the assembly of the virus and in the infection of the host cells. Whereas some functional aspects of the proteins have been studied in detail, little is known about their structure. Since X-ray analysis of these proteins appear unlikely in the near future, we chose to use a variety of computer-aided methods to improve the model for the major surface protein (SHBs). We here describe the model, discuss it in light of current results in the literature and discuss new functional implications of SHBs.

In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor.

Na+/K+-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidy1 derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylcarbonyl-6-aminocaproate (HDMA), 3-amino-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-0-succinyl-[2-(N-maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37"C, half-maximal inhibition of Na+/K+-ATPase was seen by HDMA at 0.47 pM, by ADHS at 5.8 pM, by IAD at 8 pM and by DSME at 94 pM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+-ATPase. Affinity labeling of the a subunit by 'front door' or 'back door' phosphorylation was only seen with HDMA or ADHS in the range 0.1 pM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain.Tryptic hydrolysis of the HDMA-modified Na+/K+-ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidy1 esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with CyslO4 in the H, transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-'4C]ICH,COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J. Keywords. Na'lK' -ATPase ; N-hydroxysuccinimidyl digoxigenin-3-0-methylcarbonyl-e-aminocaproate ; cardiac glycoside receptor. Na+/K'-ATPase is specifically inhibited by cardioactive steroids [l]. They bind to a specific conformation of Na+/K+-ATPase formed in the catalytic cycle during the hydrolysis of ATP [2, 31. The relatively stable complex with cardiac glycosides is formed via the forward reaction of the enzyme in the presence of Na', Mg" and ATP ('front door' phosphorylation) which forms the cardiac glycoside receptor complex I, or via phosphorylation in the presence of Mgz+ and Pi ('back door' phosphorylation) forming the cardiac glycoside receptor site 11. Enzyme. Na+/K+-ATPase, Na+/K+-transporting ATPase (EC 3.6.1.37).tein-reactive cardiac glycosides have been synthesized with the aim of localizing the cardiac glycoside-binding site [7-111. These studies demonstrated that the 40-kDa N-terminal tryptic fragment of the catalytic a su...

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine.