Germin isoforms are discrete temporal markers of wheat development. Pseudogermin is a uniquely thermostable water-soluble oligomeric protein in ungerminated embryos and like germin in germinated embryos, it is incorporated into cell walls.

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins.

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Covalently bonded and adventitious glycans in germin.

Germin was previously shown to contain covalently bonded and adventitious glycans. The object of the present investigation was to characterize the two types of glycan. The presence of N- but not O-glycans in germin is indicated by the biosynthesis of altered forms, including an unglycosylated form of germin when wheat embryos are germinated in the presence of tunicamycin. After treating the doublet of germin pentamers (G and G') from normally germinated embryos with beta-N-acetylglucosaminidase, G is converted to a form that co-migrates with G' during electrophoresis in sodium dodecyl sulfate-polyacrylamide, but G' is unaffected. This suggests that the N-glycans in G contain antennary N-acetylglucosamine but that those in G' do not. This conclusion has been confirmed and elaborated by doubly labeling G and G' in vivo with [3H]glucosamine and [35S]methionine, and by characterizing sugar-labeled glycopeptides from G and G' by gel filtration, before and after their degradation by exoglycosidases. In the context of proven structures for the complex N-glycans in other plant glycoproteins, the findings, when combined with monosaccharide analyses of G and G', permit plausible speculation about the structure of the single N-glycan that is likely present in each G monomer (GlcNAc2(Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc] and G' monomer ((Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc)). The adventitious glycans, which can be removed by phenolic extraction of germin, have a composition similar to that expected for the characteristic hemicelluloses and pectins in monocot cell walls. The possible significance of this finding is discussed in relation to our continuing efforts to define the biochemical involvements of germin. In allied studies, affinity of its N-linked glycans for concanavalin A has been used to concentrate small amounts of germin from large volumes of wheat extract and to fractionate germin from tunicamycin-treated and normally germinated wheat embryos.

Polypeptide structure of germin as deduced from cDNA sequencing.

Synthesis of a relatively rare glycoprotein (germin) signals the onset of growth in germinating wheat embryos. Germin mRNA (1075 nucleotide residues) has an 85-residue 5'-untranslated sequence, a 69-residue sequence that can encode a 23-residue signal-peptide sequence, a 603-residue sequence that can encode a 201-residue mature-protein sequence, and a 318-residue 3'-untranslated sequence that begins with a UAA-terminator codon, ends with a 63-residue polyadenylate tract, and has three polyadenylation (and other, related) signals (AAUAAN etc.). One polyadenylation signal is just 9 nucleotides from the polyadenylation site, the shortest stretch of nucleotides yet found between polyadenylation signal and site in any animal or plant mRNA. The mature-protein coding sequence in germin mRNA contains an unusually high proportion (87%) of G + C in the third positions of its codons. The amino acid sequence of germin does not have extensive internal homologies or repetitions, and it is not characterized by regions of unusually high charge density, as is nucleoplasmin, another water-soluble homopentameric protein with otherwise closely related structural properties. Germin does, however, contain a stretch of 34 uncharged amino acid residues and these may possibly mediate its homopentameric structure and its remarkable resistance to enzymic proteolysis. In view of a possible association of germin with cellular membranes, the most interesting relatedness of the germin sequence to the sequences of other proteins is an 80% homology between a decapeptide sequence in mature germin and a decapeptide sequence in Escherichia coli glycerol-3-phosphate acyltransferase. The relation of germin-gene structure to overall gene regulation during early plant growth is discussed.

Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis.

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2-45 microM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.

The modulation by serum albumin of rabbit platelet aggregation in response to exogenous sodium arachidonate.

This work presents a quantitative study of the modulation of platelet responsiveness to sodium arachidonate by serum albumin. Rabbit platelets suspended in protein-free buffer containing dextran aggregate reversibly in response to micromolar amounts of ADP and sodium arachidonate. The optimal concentration of arachidonate for aggregation response is 5 microM. Inhibition occurs at higher concentrations and is not related to thromboxane A2 formation since arachidonate inhibits ADP-induced aggregation of aspirin-treated platelets. Thin-layer chromatographic studies show that, at the high arachidonate levels sufficient to almost completely abolish platelet aggregation, the synthesis of thromboxane A2 persists. Albumin relieves the inhibition caused by excess arachidonate, whether the stimulus is arachidonate itself or ADP. This effect is due to arachidonate binding and is optimal at fatty acid/protein ratios near 4; no stimulation of platelets was observed at ratios less than 2. The optimal concentration of arachidonate for stimulation of platelet aggregation occurs in the range where albumin buffers the free arachidonate concentration most effectively, hence the extremely narrow range of total arachidonate concentrations effective for platelet response seen in the absence of albumin is enormously broadened in the presence of albumin. Albumin inhibits conversion of arachidonate to thromboxane A2 and hydroxy acids, especially at ratios of arachidonate/albumin below 10. Bilirubin (an albumin ligand) has no effect on albumin modulation of platelet response until the bilirubin/protein ratio exceeds 2. Palmitate progressively displaces arachidonate from albumin and affects the range of effective arachidonate concentrations but not the maximum response.

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells.

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.