Gly Lys Research Articles

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-napthylamide (NA) bond of benzoyl(Bz)GlyLeuArgProGlyGlyLysArg2-NA which contains two likely processing sites, ArgPro and LysArg. On the basis of the ratio of V max to K m as a measure of substrate specificity, BzGlyLeuArgProGlyGlyLys Arg2-NA is about 50-fold better than BzGlyGlyLysArg2-NA. BzLeuArg2-NA and BzGlyLeuArgProGlyGly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (BzGlyGlyLysArg2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the M r is ≈160,000 but is ≈ 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, M r ≈ 70,000.

Structural studies and two-dimensional gel electrophoresis of γ-glutamyl transpeptidase

The heterodimeric enzyme γ-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000–26,000 and isoelectric point range of 5.4–7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000–53,000 and isoelectric point range of 5.8–7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reversephase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly LysProAspHisValTyrSerArgAla, and for the first 36 residues of the light subunit with the N-terminal starting sequence ThrAlaHisLeuSerValValSerGluAsp.

Isolation and structure of a peptide toxin from the marine snail Conus magus

A 14-residue peptide toxin has been isolated from the venom of the marine snail Conus magus. Its amino acid sequence, GlyArgCysCysHisProAlaCysGly LysAsnTyrSerCysNH 2, is homologous with those of the previously described conotoxins GI, GII, and GIA from Conus geographus. The new peptide, conotoxin MI, is two to three times more active than the others, and is presumed to act as they do at the acetylcholine receptor of vertebrate neuromuscular junctions.

Solid-phase Synthesis of Substrate Model Peptides and Their Hydroxylation with Collagen Lysyl Hydroxylase

Abstract The peptides, (Ala–Lys–Gly)10–Phe, (Ile–Lys–Gly)3–Phe, and (Ile–Lys–Gly)5–Phe, were synthesized by solid-phase fragment condensation method. The contaminating shorter peptides and branched chain peptides were removed by CM-cellulose column chromatography. The peptides were useful as synthetic model substrates for collagen lysyl hydroxylase. The –Ile–Lys–Gly– units in (Ile–Lys–Gly)5–Phe showed a higher affinity to the enzyme than those in (Ile–Lys–Gly)3–Phe. A strong substrate inhibition was observed with highly basic peptide, (Ala–Lys–Gly)10–Phe.

Human complex-forming glycoprotein, heterogeneous in charge: The primary structure around the cysteine residues and characterization of a disulfide bridge

The amino acid sequence of the cysteine-containing regions of human complex-forming glycoprotein, heterogeneous in charge (protein HC) was determined by studies of the tryptic peptides of the completely reduced and radioalkylated protein. One of the cysteines was located in the amino-terminal part of the molecule at position 34: GlyProValPro ThrProProAspAsnIleGlnValGlnGluAsnPheAsn IleSerArgIleTyr GlyLysTrpTyrAsnLeuAlaIleGlySerThrCys ProLeuLys. Another was located in the carboxyl-terminal part of the molecule 14 residues from the carboxyl end: ThrMetAlaAspArgGlyGluCysValProGlyGlu GlnGluProGluProIleLeuIleProArg. The third was located somewhere in the middle of the molecule in the sequence: LysGlyValCysGluGluThrSerGlyAlaTyrGlu Lys. Diagonal map electrophoresis showed that the cysteine residue in the carboxy-terminal region was bridged to the cysteine containing sequence in the middle of the molecule. The function of the cysteine residue at position 34 remains elusive since the residue was not found on the diagonal maps. The release of cysteic acid and a small cysteic acid containing peptide after oxidation of the native protein HC molecule suggests that this cysteine residue may be involved in disulfide bridges with cysteine and small cysteine containing peptides.

ChemInform Abstract: SYNTHESIS OF CORTICOTROPIN PEPTIDES. XV. THE SYNTHESIS OF AN UNDECAPEPTIDE AND A NONAPEPTIDE DERIVATIVES RELATED TO THE AMINO ACID SEQUENCE 11‐21 OF CORTICOTROPIN

AbstractDurch schrittweisen Aufbau nach üblichen Methoden werden das Undekapeptidderivat (I) sowie das Nonapeptidderivat (II) synthetisiert.

ChemInform Abstract: SYNTHESIS OF CORTICOTROPIN PEPTIDES. X. THE SYNTHESIS OF ACTH(1‐18)‐OH, ACTH(1‐18)‐NH2 AND ACTH(1‐19)‐NH2

AbstractDie Synthese der Oktadekapeptide (Ia) und (Ib), die den Aminosäuresequenzen 1‐18 von Corticotropin (ACTH) entsprechen, erfolgt durch Kupplung des geschützten N‐Hydroxysuccinimidesters des ‐ bekannten ‐ N‐terminalen Dekapeptids mit den ‐ ebenfalls bekannten ‐ entsprechenden Oktapeptidderivaten, Schutzgruppenabspaltung und Reinigung.

Synthesis of Corticotropin Peptides. XV. The Synthesis of an Undecapeptide and a Nonapeptide Derivatives Related to the Amino Acid Sequence 11–21 of Corticotropin

Abstract An undecapeptide derivative, Z–Lys(Mhoc)–Pro–Val–Gly–Lys(Mhoc)–Lys(Mhoc)–Arg–Arg–Pro–Val–Lys–(Mhoc)–NHNH2, corresponding to the amino acid sequence 11–21 of corticotropin (ACTH)is synthesized. In the course of the synthesis a key intermediate H–Arg(NO2)–Arg(NO2)–Pro–OH is derived from Z–Arg(NO2)–Arg–(NO2)–Pro–OBzl by the treatment with hydrogen bromide in acetic acid. This procedure eliminates the danger of racemization associated with the use of alkali for saponification. The synthesis is also described of a nonapeptide derivative, Z–Lys(Mhoc)–Pro–Val–Gly–Lys(Mhoc)–Lys(Mhoc)–Arg(NO2)–Arg(NO2)–Pro–OH (amino acid sequence 11–19 of ACTH), in which a new protecting group, 9-methyl-9-fluorenyloxycarbonyl (Mfoc), is utilized for the temporary protection of an α-amino function.

Synthesis of Corticotropin Peptides. X. The Synthesis of ACTH(1–18)–OH, ACTH(1–18)–NH2 and ACTH(1–19)–NH2

Abstract The final coupling, deprotection and purification processes for the synthesis of octadecapeptides, H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–OH(I) and H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–NH2 (II), corresponding to the first eighteen amino acid residues of corticotropin (ACTH) are described, in which the N-hydroxysuccinimide ester of the N-terminal decapeptide derivative is coupled with the C-terminal octapeptide and octapeptide amide derivatives to give the two protected end products. The same decapeptide active ester is also utilized to the synthesis of a nonadecapeptide, H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–Pro–NH2 (III), identical with the first nineteen amino acid residues of ACTH. The in vivo steroidogenic potencies of the synthetic peptides are compared with that of the synthetic human ACTH (αh-ACTH) as reference to show that the relative potencies of I, II, and III vs. αh-ACTH are 0.14, 0.48, and 0.47, respectively, as estimated on a molar basis.

Improved Solid-Phase Synthesis of Tryptophan-Containing Peptides. II. Use of Nα-t-Butyloxycarbonyl-Ni-formyltryptophan

Abstract Ni-Formyltryptophan has proved to be more resistant than tryptophan against oxidation mediated by hydrogen chloride in acetic acid. Nα-t-Butyloxycarbonyl-Ni-formyltryptophan has been synthesized via nonaqueous acylation reaction and used for the solid-phase synthesis of tryptophan-containing peptides, in combination with hydrogen chloride in formic acid as the reagent for cleavage of the t-butyloxycarbonyl group. The validity of the method has been demonstrated, on the basis of several criteria, in the synthesis of the tryptophan-containing octapeptide, Lys–Gly–Val–Leu–Ala–Gly–Trp–Leu.

Motilin, a gastric motor activity stimulating polypeptide: the complete amino acid sequence.

Porcine motilin is a 22 amino acid residue polypeptide with the amino acid sequence Phe–Val–Pro–Ile–Phe–Thr–Tyr–Gly–Glu–Leu–Gln–Arg–Met–Glu–Glu–Lys–Glu–Arg–Asn–Lys–Gly–Gln. The calculated molecular weight is 2700.

Amino acid sequences in the β-chains of adult bovine hemoglobins C-Rhodesia and D-Zambia

Differences in adult bovine hemoglobins C-Rhodesia and D-Zambia reside in the β chains. The substitutions (C → D) occur at residue 20 (Asp → Gly), residue 43 (Ser → Thr), and residue 131 (Gln → Lys) of the 145-residue chain. The changes are unrelated to the previously determined substitutions between bovine hemoglobins A and B [(1967) Arch. Biochem. Biophys. 120, 124]. The differences in the four types of chain are Residue No. 15 18 20 43 119 131 β A Gly Lys Asp Ser Lys Lys β B Ser His Asp Ser Asn Lys β C Gly Lys Asp Ser Lys Gln β D Gly Lys Gly Thr Lys Lys Thus, as compared to bovine hemoglobin A, the changes between pairs are AB-3, AC-1, and AD-2. Bovine hemoglobin C denatures more readily in alkali than do the others and dissociates at a lower alkaline pH. The data add to previously observed multiple differences between animal hemoglobin variants which are in contrast to the case in human variants. Structural and genetic implications are discussed.

The Synthesis and Reaction of Monohalogeno-t-butyloxycarbonylamino Acids

Abstract Monohalogeno-t-butyloxycarbonylamino acids (XBOC-amino acids) were synthesized by the use of the corresponding chloroformates in a basic aqueous solution. Several dipeptides containing XBOC-amino acid were also prepared. The cleavage of the XBOC group was successfully carried out not only under some acidic conditions, but also by heating in methanol or ethanol. In order to demonstrate the usefulness of this group in peptide synthesis, Z–Gly–Lys(Z)–Gly–OEt was synthesized via BrBOC–Lys(Z)–Gly–OEt, which had been treated by hot ethanol. Side reaction during the synthesis of XBOC-amino acids will also be described.

A gastric inhibitory polypeptide. II. The complete amino acid sequence.

Porcine gastric inhibitory polypeptide is a 43 amino acid residue polypeptide with the amino acid sequence Tyr–Ala–Glu–Gly–Thr–Phe–Ile–Ser–Asp–Tyr–Ser–Ile–Ala–Met–Asp–Lys–Ile–Arg–Gln–Gln–Asp–Phe–Val–Asn–Trp–Leu–Leu–Ala–Gln–Gln–Lys–Gly–Lys–Lys–Ser–Asp–Trp–Lys–His–Asn–Ile–Thr–Gln. Fifteen of the first 26 amino acids occur in the same position as they do in porcine glucagon, and nine of the first 26 in the same position as in porcine secretin. The calculated molecular weight of the polypeptide is 5105.

A gastric inhibitory polypeptide. I. The amino acid composition and the tryptic peptides.

The partial structure of a polypeptide with inhibitory activity for gastric secretion has been found to be Tyr–(Ala2, Asx, Glx, Gly, Ile2, Phe, Ser2, Thr, Tyr)–Met–Asp–Lys–Ile–Arg–Gln–(Ala, Asx2, Glx2–3, Leu, Phe, Val, Trp)–Lys–Gly–Lys–Lys–Ser–Asp–Trp–Lys–His–(Asx, Ile, Thr, Gln).