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J T Gafford

Publications and source records attributed to J T Gafford.

10 recordsLinked to original sources
PubMed1985

Neutral metalloendopeptidase in human male genital tract. Comparison to angiotensin I-converting enzyme.

High concentrations of neutral metalloendopeptidase (NEP) (enkephalinase) were found in human male genital tract immunohistochemically and by enzyme activity assays, and its distribution was compared with that of angiotensin-converting enzyme (ACE) (kininase II). Whereas the two enzymes colocalize on the luminal aspect of proximal tubular epithelium and are not found elsewhere in the nephron, their distribution in the male genitalia is different. Seminal fluid is rich in NEP and ACE, but after ultracentrifugation ACE remains soluble while NEP sediments. NEP activity is low in testicular homogenate but high in the particulate fraction of epididymides and prostates. ACE, on the other hand, is active in the particulate fraction of testes and in the soluble fraction of epididymides and prostates. Prostatic NEP had a slightly higher molecular weight than the renal NEP, which was reduced by neuraminidase in electroblotting. Testicular and seminal plasma ACE also had a slightly higher molecular weight than the purified renal enzyme (150,000), probably caused by removal of an "anchor" peptide during purification. In the prostate, NEP was found by three different immunohistochemical techniques in luminal epithelial cells and in lumina. The function of NEP in the genital tract may be related to sperm maturation and proacrosin activation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Human kidney "enkephalinase", a neutral metalloendopeptidase that cleaves active peptides.

After extracting converting enzyme from a membrane fraction of homogenized human kidney, "enkephalinase" activity was solubilized with Triton X-100. Ion-exchange chromatography resolved two peaks of the "enkephalinase" activity, both of which cleaved Leu5-enkephalin at the Gly3-Phe4 bond. The major "enkephalinase" form was purified 1140-fold to homogeneity with a 14% yield. This homogeneous "enkephalinase" had a specific activity of 46 mumol min-1 mg-1 with Leu5-enkephalin as substrate. The purified enzyme, in addition to hydrolyzing Leu5-enkephalin, cleaved synthetic substrates with protected N- and C-terminal ends. On the basis of the specificity of the enzyme and its inhibition by chelating agents, human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity. The activity of this neutral endopeptidase with several biologically active peptides was compared to that of homogeneous human kidney converting enzyme. Both enzymes inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors. Comparison of hydrolysis of bradykinin with that of its protected C-terminal peptide indicated that the neutral endopeptidase is more active toward the larger substrate than is converting enzyme. Although the neutral endopeptidase did not convert angiotensin I to II, it did hydrolyze angiotensin I at Pro7-Phe8 and inactivate angiotensin II by cleavage at the Tyr4-Ile5 bond.

Angiotensins↗
PubMed1983

Novel substrates for angiotensin I converting enzyme.

Homogenous human angiotensin converting enzyme (EC 3.4.15.1) cleaves dipeptides from the C-terminus of substrates containing a free carboxyl group. In this study we demonstrate that peptides containing a C-terminal nitrobenzylamine are also cleaved by the enzyme. The hydrolysis of these substrates is inhibited by the specific converting enzyme inhibitors captopril and MK421 as well as by anti-converting enzyme antibody. Sodium chloride accelerates the rate of hydrolysis forty-fold. The product of the reaction, an amino acid nitrobenzylamide, was identified by thin layer chromatography and high performance liquid chromatography. These results suggest that the carboxyl group is not an absolute requirement for substrate hydrolysis.

Amino Acid Sequence↗
PubMed1983

Human converting enzyme.

Angiotensin I converting enzyme (kininase II, peptidyl dipeptidase, ACE) was purified by reverse immunoadsorption from a membrane fraction of the human kidney. ACE is very likely a transmembrane peptidase. Treatment of the membrane-bound enzyme with trypsin releases a low mol. wt. fragment (greater than 10,000), which is probably the anchor peptide inserted into the plasma membrane. Antibody to ACE was used to localize it in the CNS where it is bound to plasma membrane of neuroepithelial cells in structures such as the globus pallidus or substantia nigra. Radioimmunoassay indicated that ACEs of endothelial, epithelial and neuroepithelial origin are immunologically identical. Direct radioimmunoassay also showed that there is a strong negative correlation between plasma enzyme level and pulmonary diffusing capacity of sarcoid patients. Finally, in addition to various peptides, homogeneous human ACE cleaves fluorogenic substrates where the C-terminal amino acid is replaced with nitrobenzylamine.

Cell Membrane↗
PubMed1977

Tunicamycin inhibits GlcNAc-lipid formation in plants.

Previous studies from this laboratory (Forsee, W. T., and Elbein, A. D. (1975) J. Biol. Chem. 250, 9283-9293; Forsee, W. T., Valkovich, G., and Elbein, A. D. (1976) Arch Biochem. Biophys. 174, 469-479) have shown that particulate extracts from cotton fibers and mung been seedlings catalyze the transfer of mannose from GDP-[14C]mannose and GlcNAc from UDP-[3H]GlcNAc into lipid-linked saccharides, Concentrations of tunicamycin of 5 microgram/ml or higher inhibit the incorporation of GlcNAc into GlcNAc-pyrophosphoryl-polyprenol but this antibiotic, even at 500 microgram/ml, had no effect on the synthesis of mannosyl-phosphoryldolichol. Tunicamycin also caused a slight inhibition in the incorporation of mannose into lipid-linked oligosaccharides. The concentration of tunicamycin necessary for inhibition was dependent on the amount of particulate enzyme in the incubations.

Acetylglucosamine↗
PubMedDate not supplied

Enzymes in placental microvilli: angiotensin I converting enzyme, angiotensinase A, carboxypeptidase, and neutral endopeptidase ("enkephalinase").

Microvilli from human placental syncytiotrophoblast are rich in angiotensin I converting enzyme (ACE), aminopeptidase A, a carboxypeptidase N-like enzyme, and a neutral endopeptidase (NEP). The specific activities of these enzymes were enhanced in microvillus-enriched fractions obtained by differential centrifugation: Purified microvilli were isolated in a discontinuous sucrose gradient. The placental microvilli hydrolyzed angiotensin II, vasopressin and oxytocin as shown by high pressure liquid chromatography. The inhibitors, bestatin, phosphoramidon, and o-phenanthroline, established the specificity of the enzymes. Aminopeptidase A (angiotensinase A) cleaved angiotensin II to angiotensin III and Asp1. NEP from placenta and from human kidney hydrolyzed oxytocin at the Pro7-Leu8 bond to yield oxytocin 1-7 and leucyl-glycine amide, but did not hydrolyze vasopressin. Vasopressin was cleaved by aminopeptidases in the placental membranes. On electroblotting placental NEP appeared as a double band with a molecular weight slightly higher than the 90,000 of the purified kidney enzyme. Neuraminidase treatment reduced the molecular weight of the placental enzyme to approximately 90,000, indicating that it contains a large amount of sialic acid. The microvilli of human placenta are thus rich in enzymes that may regulate passage of peptides at the maternal-fetal interface.

Aminopeptidases↗
PubMedDate not supplied

Inhibition of human converting enzyme in vitro by a novel tripeptide analog.

We have studied inhibition of homogeneous human converting enzyme by a new inhibitor, a ketomethylene derivative of the blocked tripeptide substrate, Bz-Phe-Gly-Pro (ketoACE). KetoACE inhibited the hydrolysis of Hip-His-Leu and Hip-Phe-Arg at different concentrations (I50 values were 4 X 10(-8) M and 2 X 10(-7) M, respectively). Kinetic studies indicated that ketoACE inhibits the hydrolysis of both substrates by a similar, non-competitive mechanism. At the lowest enzyme concentration tested, using 3H-Hip-Gly-Gly as substrate, the I50 of ketoACE was 6 X 10(-9) M. KetoACE protected a functional tyrosine residue in the active site of human converting enzyme from modification with N-acetylimidazole. It is proposed that there are alternate (hydrophobic) binding sites for both inhibitors and substrates in the active site of human converting enzyme. It should be possible to develop other high-affinity inhibitors of this class that bind to hydrophobic sites and do not require metal binding via a sulfhydryl group.

Angiotensin-Converting Enzyme Inhibitors↗