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Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general. [ABSTRACT FROM AUTHOR]

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number . [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of turnip peroxidase TP 7, the principal isoperoxidase during winter in turnip, Brassica napus L., variety Blanc dur d'hiver, has been determined by sequence analysis of cyanogen bromide fragments and of tryptic peptides. The turnip peroxidase TP 7 enzyme is composed of 296 amino acids, one hemin group and one neutral carbohydrate side chain attached through asparagine. The molecular weight of the polypeptide part is 31 060, and including heroin and carbohydrate the molecular weight of the native enzyme is close to 33 400. The isoelectric point of turnip peroxidase TP 7 is 11.6. Comparison of turnip peroxidase TP 7 and horseradish peroxidase HRP C shows that they contain four similarly located disulfide bridges and have pyrrolidone carboxylyl N termini. Their common evolutionary origin is distant as their amino acid sequences are only 49% identical. Furthermore, turnip peroxidase TP 7 differs significantly from three other isoperoxidases of turnip root, turnip peroxidases TP 1, TP 2 and TP 3, and from horseradish peroxidase HRP C in its physico-chemical and enzymatic properties, and its pronounced season-dependent appearance. All these differences of turnip peroxidase TP 7 and of the others suggest they serve separate biological functions. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a heroin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3. Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion. [ABSTRACT FROM AUTHOR]

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences near the peroxidase heme prostetic group, in the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heine, and 34 residues around the probably distally located histidine, and compare them with the histidine-containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3 and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physico-chemical and enzymic properties. Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, P2 and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site. [ABSTRACT FROM AUTHOR]

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a singlebase mutation, changing an asparagine residue to a lysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heine prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail. [ABSTRACT FROM AUTHOR]

Abstract: Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide. [Copyright &y& Elsevier]