In order to elucidate the epidemiological importance of hemorrhagic fever with renal syndrome in Germany, the prevalence of antibodies against hantaviruses was determined in 13,358 sera from residents of various geographic regions, 1,284 sera from occupational risk groups and 287 sera from chronic hemodialysis patients. Serological investigations were performed using a highly specific transferable solid phase enzyme immunoassay based on the recombinant nucleocapsid proteins of a Hantaan and a Puumala serotype strain. The overall antibody prevalence was found to be 1.68%. In the serum panels from western and southern Germany, it was determined to be 1.83% on average in contrast to only 0.8% in the panel from eastern Germany. An endemic focus revealing an antibody prevalence of 3.12% was detected in a low-mountain area called Suebian Alb, which is located in the federal state of Baden-Württemberg. Occupational risk groups and a group of chronic hemodialysis patients showed a significantly elevated antibody prevalence ranging from 3.3% to 10%. The Puumala serotype was found to be the prevailing virus, but the percentage of sera predominantly recognizing the Hantaan nucleocapsid protein increased towards the south and the east and was significantly elevated in dialysis patients.

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativu L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDSPAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydrylendopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna tnungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of I-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.Keywords: triggering cysteine endopeptidase ; seed germination; Vicia sativa L. ; endoplasmic reticulum retention signal.The beginning of storage-protein degradation is indicated by slight mobility changes of globulins from cotyledon extracts of germinating dicotyledonous seeds on electrophoresis (Shutov and Vaintraub, 1973;Lichtenfeld et al., 1979Lichtenfeld et al., , 1981Boylan and Sussex, 1987; Dunaevsky and Belozersky, 1989). During this first stage of breakdown the holoproteins remain assembled, which is reflected by nearly unchanged positions in the sucrosedensity gradient after centrifugation and in gels after electrophoresis under non-denaturing conditions. Only small trichloroacetic-acid-soluble peptides are lost as the result of limited proteolysis (Shutov and Vaintraub, 1987). These first modifications are thought to be catalyzed either by stored endopeptidases that have to be activated just after imbibition, as in buckwheat (Elpidino et al., 1991), or by newly formed endopeptidases, as assumed for the majority of legumes (Chrispeels et al., 1976; Lichtenfeld et al., 1979;Muntz et al., 1985; Shutov and Vaintraub, 1987). Only after 3-6 days depending on the species and germination conditions, a second stage with rapid storageglobulin degradation begins, accompanied by strong increa...

Proteinase B, an asparagine‐specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C‐terminal side of Asn residues. The octapaptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross‐reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA clones encoding proteinase B precursor have been obtained on the basis of the N‐terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA‐derived amino acid sequence of a vacuolar Asn‐specific enzyme from the developing castor bean endosperm which is thought to catalyze the post‐translational processing of pro‐proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern‐blot analyses suggested that there are at least two genes for proteinase B.

Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapaptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development.cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asnspecific enzyme from the developing castor bean endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.