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A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27–63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 µg·g−1 body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology. [ABSTRACT FROM AUTHOR]

The primary structure of glycolate oxidase from spinach has been determined. Six different types of peptide digest were investigated, utilizing CNBr, proteolytic enzymes, and chemical modifications to change a specificity of cleavage. In total, 90 peptides were purified and analyzed. The studies were aimed at correlation with crystallographic analysis of the same protein carried through in parallel and with eDNA studies which utilized initially determined amino acid sequences for synthesis of oligonucleotide probes. Continuous comparisons with the results from the crystallographic studies helped at an early stage to secure peptide overlaps, at the same time as the peptide data secured residue assignments in the electron density maps. In the end, all data agree and regions from all parts of the molecule have been checked by independent methods of analysis. The primary structure establishes the type of N-terminal post-translational processing, and yields information on segments not fully defined in electron density maps. Combined, the chemical, crystallographic, and eDNA data give extensive reliability. The peptide analysis shows that the N-terminus is blocked by acylation of the initiator methionine, which is in a primary structure typical for non-removal of the methionine in the processing events of the nascent protein chain. The molecule is comparatively rich in menthionine and some other generally less common residues, but has only one cysteine residue and no extensive hydrophobic segment. An amino acid sequence homology with flavocytochrome b2 from yeast, as expected from known similarities in tertiary structure, is observed (33% residue identities). [ABSTRACT FROM AUTHOR]

The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The [14C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre-fractionation and reverse-phase high-performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and the use of liquid-phase sequencer degradations. All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met-Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354-residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl-blocked, the C terminus is formed by a prohne residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at postion 43). Similarities to 'long' alcohol dehydrogenases have functional implications. [ABSTRACT FROM AUTHOR]