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G Fleminger

Publications and source records attributed to G Fleminger.

43 records · Page 3Linked to original sources
PubMed1983

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases.

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.

Amino Acid Sequence↗
PubMed1983

Synthesis of adrenal peptide E and some of its biological activities.

A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.

Amino Acid Sequence↗
PubMed1983

Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.

Animals↗
PubMed1982

Fluorogenic substrates for bacterial aminopeptidase P and its analogs detected in human serum and calf lung.

A sensitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized. Phe(NO2)-Pro-HN-CH2-CH2-NH-ABz (substrate I) and Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-ABz (substrate II), in which the Phe(NO2) group (rho-nitro-L-phenylalanyl) quenches the fluorescence of the ABz group (omicron-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing rho-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively amino-peptidase P-like activity in the human serum and calf-lung extracts. Substrate II was shown to be specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro-HN-CH2-CH2-NH-ABz (Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf-lung enzymes respectively. The enzyme activity was only slightly dependent on pH in the range 7.0-8.4.

Aminopeptidases↗
PubMedDate not supplied

Functional conformations of calmodulin: I. Preparation and characterization of a conformational specific anti-bovine calmodulin monoclonal antibody.

Calmodulin, similarly to many other Ca(2+)-activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to its high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAM1, CAM2 and CAM4) were purified and characterized. MAb CAM1 was identified as an IgG1 while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAM1 binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAM1 was found to be especially sensitive to the conformational state of calmodulin in the presence of Ca2+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca2+ removal. In addition mAb CAM1 failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM1 and CAM4 share common epitopes with the above molecules.

Animals↗