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Reports on a study in which gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. Reverse-phase high-performance liquid chromatography; Screening of an enriched cDNA library for the β-GTH genes using two synthetic oligonucleotides; Effect of testosterone implantation on pituitary PTH and β-GTH mRNA; Corresponding increase in both the pituitary GTH and mRNA levels.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem. 267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 1933) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) thru differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-L-isoaspartic acid O-methyltransferase (PIMT; EC2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Serl6) of this 28-residue peptide, a time-dependent mobility shift from t[SUBR] ≈ 34 min to ≈ 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the t[SUBR] ≈ 34 min species decreased with a biphasic time-course with t[SUB0.5]-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ≈ 1.5 days in 150 mM Tris/HC1, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial β-isomerization... [ABSTRACT FROM AUTHOR]

Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidylpeptidase IV as auxilliary enzyme, was used m monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. [ABSTRACT FROM AUTHOR]

Five tridecapeptides have been identified from the central nervous system of the pond snail, Lvmnaea stagnalis. The sequences are Pro-Xaa-Asp-Arg-Ile-Ser-Yaa-Ser-A1a-Phe-Ser-ASp- Phe NH2. where Xaa is either Tyr or Phe and Yaa either Asn, Ser or Gly. The peptides are named lymnaDFamides to acknowledge identity with the C-terminal dipeptide of the mammalian neuropep- tides. cholecystokinin (CCK) and gastrin. They were detected by an antiserum that recognizes the biologically active C-termini of cholecystokinin and gastrin. LymnaDFamide-1 (Xaa = Tyr and Yaa = Asn had no effect on trout gallbladder, which responds equally to CCK and gastrin. We propose that the IymnaDFamides belong to an Asp-Phe-amide superfamily. which includes CCK and gastrin. and suggest that the widespread CCK/gastrin immunoreactivity in invertebrates is due to peptides belonging to such a superfamily. [ABSTRACT FROM AUTHOR]

The major peptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea) has been extracted and purified by reverse-phase HPLC. This neuropeptide exhibited a high hyperglycemic activity' and was therefore named A. vulgare crustacean hyperglycemic hormone (Arv-CHH). Its average molecular mass measured by mass spectrometry was 8729.3 Da. Its complete amino acid sequence was determined by a combination of Edman degradation and mass spectrometry. The N-terminal amino acid was found to be unblocked, the C-terminal residue was found amidated and none of the other 72 residues was affected by any post-translational modification. Disulfide bond assignment was made unambigously by mass spectrometry and Edman degradation was performed on peptides produced by enzymatic cleavage. Relationships with other, similar neuropeptides from decapod sinus glands are discussed. [ABSTRACT FROM AUTHOR]

The class I of the high mobility group (HMG) proteins is formed by phosphoproteins which are associated with AT-rich DNA sequences in the nucleus. Three HMGI proteins have previously been described in proliferating rodent cells (HMG Y, HMG I and HMGI-C). All three proteins exhibit microheterogeneity. The microheterogeneity of mouse HMG Y has been investigated in detail and shown to be due to phosphorylation of the protein which is sensitive to alkaline-phosphatase treatment. HMG I is similarly modified. Human cells have up to now only been found to contain HMG Y and HMG I. A search for the third protein, HMGI-C, in human cells was carried out and the protein was found in a hepatoma cell line, but not in normal or transformed T-cells. This HMGI-C protein was found to be modified by phosphorylation, part of which was found to be phosphatase insensitive. An unexpected additional finding in this study was that human cells contain two HMG17 proteins which differ in their N-terminal primary sequences. [ABSTRACT FROM AUTHOR]

Secretory actin-binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206-210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13506 and pyroglutamic acid as the N-terminal residue. A single N-glycosidic carbohydrate moiety is located at Ash77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37-Cys63 and Cys61-Cys95. Sequence comparison revealed a high degree of similarity with a 14-kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin-inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues. Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function. [ABSTRACT FROM AUTHOR]

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium · HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography.Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4'-dimaleimidestilbene 2,2'-disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS-PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide-binding sites per dimer was determined by fluorimetric titrations using pyridoxyl-ATP, a strong competitive inhibitor with respect to ATP. The ATP analog binds with a Kd = 5 µM to each nucleotide site of the dimeric enzyme. The mode of binding hyridoxyl-ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the single polypaptide chain of bovine plasminogen (786 residues, Mr 88092) was determined. Cleavage with CNBr yielded 13 fragments of which six originated from cleavage sites different from human plasminogen. Digestion with elastase gave three major fragments: kringles (1+2+3) and kringle 4, both with intact lysine binding sites, and mini-plasminogen. Subfragmentation was achieved mainly with 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine (BNPS-skatole), Staphylococcus aureus V8 protease and trypsin. The sequences of fragments which were determined by automated Edman degradation, were aligned with overlapping sequences, or, in a few instances, by homology with the known sequence of human plasminogen. Sequence comparison with the human protein showed varying degrees of homology in the different functional and structural domains. The overall identity (78%) is practically the same as that found in those regions corresponding to the heavy (79%) and the light chain (80%) of plasmin. The average degree of identity among the kringles is 83%. Outside the kringle structures the extent of identity decreases, to 65% in the N-terminal region and to about 50% in the connecting strands between the kringles except for the strand between kringles 2 and 3, where only one out of 12 residues is exchanged. The results reported show that bovine plasminogen apparently contains the same structural and functional domains as human plasminogen. Bovine plasminogen also contains two carbohydrate moieties, The only partially substituted N-glycosidic site, Asn289, corresponds to partially glycosylated Asn288 in human plasminogen, whereas the O-glycosidic site of the human sequence, Thr345, is shifted to Ser339 in bovine plasminogen. [ABSTRACT FROM AUTHOR]

Characterizes the biological properties of enzyme-resistant analogues of substance P. Enzyme specificity; Enkephalin analogue; Synthesis of undecapeptides and heptapeptides; Binding to receptor sites; Protection of peptide bonds on the carboxyl side of residues 6,7 and 8 of substance P.