Protein derivatives exhibiting aerating and whiping properties are produced by the action of a proteolytic enzyme on a suitable protein substrate, viz., wheat gluten, soy protein or casein. Various proteolytic enzymes can be utilized in the manufacture of the modified protein derivatives, but those enzymes displaying maximum proteolytic activity in the acid pH range produce whipping proteins with optimum aerating functionality and maximum stability characteristcs. Reaction parameters for the hydrolysis include: type of enzyme, enzyme concentration, temperature, pH and time of reaction. By careful control of these variables, modified proteins with unique and exceptional whipping properties can be produced. Enzyme‐modified whipping proteins are bland, light cream‐colored, spray‐dried powders, soluble in hot or cold water, and functional over the entire pH range. Depending upon the source, protein and conditions of manufacture, products can be produced with a protein content ranging from as low as 50% to as high as 85%, and a whipping functionality equal to or twice that of egg albumen. Unlike egg albumen, these products do not coagulate to any appreciable degree when heated, and depending upon the specific end use of the product, this property may be either an advantage or disadvantage.

A particulate translation system isolated from the yeast Succhuromyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-a-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-a-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-a-Fo) differing in molecular mass from a cytosolically located, protease-K-sensitive a-factor polypeptide (Pp-tx-FcyJ by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated a-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product ppa-Fcyt but larger than the membrane-specific pp-a-Fo.Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors ( e g 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-a-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase I1 and a trimming mannosidase, which process the prepro-a-factor glycans down to the (Man)8(GlcNAc)z stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes. N-Glycosylation of proteins is assumed to be a cotranslational event which occurs shortly after or during the translocation of the nascent polypeptide chain from its cytosolic site of synthesis into the luminal space of the endoplasmic reticulum. This reaction is catalysed by membrane-bound N-glycosyltransferases which transfer the (Glc)3(Man)9(GlcNAc)z oligosaccharide from dolichyl diphosphate onto specific asparagine residues within the polypeptide chain. A necessary, though in itself not sufficient,

When programmed with yeast prepro-alpha-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-alpha-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated alpha-factor precursor (pp-alpha-F3, 27.5 kDa). Glycosylation of the membrane specific pp-alpha-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-alpha-F0), whereas the primary translation product pp-alpha-Fcyt is not affected. Likewise, only the glycosylated pp-alpha-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between pp-alpha-F0 and pp-alpha-Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-alpha-Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-alpha-F3 polypeptide indicates that its oligosaccharide chains are processed to presumably Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxyproline-containing peptides of different length and amino acid sequence have been used to demonstrate UDP-L-arabinose-hydroxyproline-O-glycosyltransferases in a crude microsomal fraction from the green alga Volvox carteri. The formation of 0-glycosidic linkages by transfer of UDP-activated arabinose to the side chain of hydroxyproline was concluded from the resistance of the glycopeptides under the basic conditions of B-elimination and their susceptibility to hydrolysis by trifluoroacetic acid. This treatment yielded arabinose as the only cleavage product. Arabinose transfer to the various peptide substrates was found to be stimulated by low concentrations of detergent, to require divalent cations and to proceed optimally at pH values around 7.0, The smallest arabinose acceptor peptide was the tripeptide Tyr-Hyp-Lys. The glycosyl acceptor effectivity increased with increasing numbers of repeated hydroxyproline residues, suggesting that hydroxyproline clusters critically affect substrate recognition by the Volvox transferase(s).Hydroxyproline cluster; 0-Arabinosylation; UDP-L-arabinose-hydroxyproline-O-glycosyltransferase; (I/olvox carteri)