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L D Fricker

Publications and source records attributed to L D Fricker.

79 records · Page 5Linked to original sources
PubMed1984

A fluorometric assay for angiotensin-converting enzyme activity.

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.

Animals↗
PubMed1984

Purification and characterization of a membrane-bound enkephalin-forming carboxypeptidase, "enkephalin convertase".

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.

Adrenal Medulla↗
PubMed1983

Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase.

Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.

Adrenal Glands↗
PubMed1983

Enkephalin convertase: potent, selective, and irreversible inhibitors.

Biproduct analogs of lysine and arginine are potent inhibitors of enkephalin convertase, a carboxypeptidase B-like enzyme which appears to be physiologically associated with enkephalin biosynthesis. The most effective derivatives are guanidinoethylmercaptosuccinic acid and guanidinopropylsuccinic acid with respective Ki's of 8.8 and 7.5 nM. These dicarboxylic acid analogs of arginine are several hundred fold more potent towards enkephalin convertase than towards carboxypeptidase B or N. Kinetic analysis indicates the pure competitive nature of the inhibition. Bromoacetyl-D-arginine, an irreversible inhibitor of carboxypeptidase B and N, is also an efficient irreversible inhibitor of enkephalin convertase.

3-Mercaptopropionic Acid↗
PubMed1982

Enkephalin convertase: purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules.

A specific carboxypeptidase that converts enkephalin precursors into enkephalin in adrenal chromaffin granules has been purified and characterized. In the adrenal this enzyme, designated enkephalin convertase, is uniquely localized to the chromaffin granules, which contain enkephalin and precursor peptides. Enkephalin convertase is markedly stimulated by CoCl2 and inhibited by EDTA or 1,10-phenanthroline, unlike the lysosomal carboxypeptidase. The purified enzyme has a high affinity for the hexapeptides [Met5]- and [Leu5]enkephalin-Arg6 (51 and 83 microM, respectively) and a somewhat lower affinity for the hexapeptides [Met5]- and [Leu5]enkephalin-Lys6 (195 and 174 microM). Brain enkephalin convertase shows 10-fold regional variations, unlike other carboxypeptidases, which are uniformly distributed. Enkephalin convertase appears to be associated selectively and physiologically with biosynthesis of the enkephalins.

Adrenal Medulla↗
PubMedDate not supplied

Cloning and sequence analysis of cDNA for bovine carboxypeptidase E.

Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules. A similar enzyme is present in many brain regions and in purified secretory granules from rat pituitary and rat insulinoma. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form, which differ slightly in relative molecular mass (Mr). Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides.

Amino Acid Sequence↗