Cystinyl Aminopeptidase Research Articles

Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [ 125I]Ang IV binding and catalytic activity

The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT 4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [ 125 I ]Ang IV and by measuring the cleavage of l-leucine- p-nitroanilide. The equilibrium dissociation constant of [ 125 I ]Ang IV in saturation binding studies ( K D = 0.90 nM) was similar to the value ( K D = 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the “AT 4 receptor” ligands (Ang IV > divalinal 1-Ang IV ∼ LVV-hemorphin-7 ∼ LVV-hemorphin-6 > Ang (3–7) > Ang III > Ang (4–8)) but not by AT 1/AT 2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other “AT 4 receptor” ligands (Ang IV > Ang III ∼ divalinal 1-Ang IV ∼ Ang (3–7) ∼ LVV-hemorphin-7 > Ang (4–8) ∼ LVV-hemorphin-6). High affinity binding of [ 125 I ]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.

Ectodomain Shedding of Cystinyl Aminopeptidase from Human Placental Membranes

Cystinyl aminopeptidase (CAP; EC 3.4.11.3) is an integral protein of the placental membrane that is also found in a soluble form in maternal serum during pregnancy. CAP was found to be shed from human placental membranes in a temperature- and time-dependent process. The released form of CAP was hydrophilic as assessed by phase separation in Triton X-114 and high-speed centrifugation. This ectodomain shedding of CAP was inhibited by the hydroxamic acid-based compounds marimastat and BB3103. The inhibition profile for the shedding of CAP was distinct to that for the release of angiotensin converting enzyme, implicating the involvement of distinct zinc metallosecretases in the shedding of these two proteins. These results have implications for our understanding of the mechanism underlying the reduction in serum levels of CAP observed in certain pregnancy-related disorders, such as pre-eclampsia.

Bestatin as an experimental tool in mammals.

Bestatin, an antibiotic of microbial origin, is a potent inhibitor of some, but not all aminopeptidases. It can be administered, with low toxicity, to cultured cells, intact animals and humans. It has become a useful tool in elucidating the physiological role of some mammalian exopeptidases in the regulation of the immune system, in the growth of tumors and their invasion of surrounding tissues, and in the degradation of cellular proteins. Bestatin-sensitive enzymes play important roles in the digestion and absorption of peptides in the brush border of the intestine and the kidney, in the reproductive system, and in the metabolism of opioid peptides and leukotrienes. Aminopeptidase N emerges as the major target for the effects of bestatin on the immune system and some of its effects on tumor growth and the endometrium. It is also the major bestatin-sensitive enzyme involved in the degradation of oligopeptides on the surface of intestine and kidney brush borders, and the inactivation of enkephalins in the brain. Bestatin-sensitive cytosolic exopeptidases are important in the degradation to amino acids of di- and tripeptides generated in most cells by cellular protein degradation, as well as those absorbed through the brush border of intestine and kidney. Inhibition of one of these exopeptidases, cytosol alanine aminopeptidase, results in apoptosis. Bestatin-sensitive cystinyl aminopeptidase is abundant in placenta. Two bestatin-sensitive enzymes, aminopeptidase B and nardilysin, are particularly abundant in late spermatids. Finally bestatin-sensitive LTA4 hydrolase generates the potent chemotactic agent, LTB4.

Immunoaffinity Purification and Characterization of Native Placental Leucine Aminopeptidase/oxytocinase from Human Placenta

cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopeptidase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is membrane-bound and that the soluble form of this enzyme, found in maternal sera, is most likely derived from the native form. The presence of the different forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogeneity, 5039-fold within 4h, by immunoaffinity chromatography. Zn2+and Cu2+strongly inhibited the enzyme but Ca2+did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-LAP, protein having 83 per cent of l -methionine insensitive Leu-p-nitroanilide cleaving activity, was immunoprecipitated from the microsomal fraction of human placenta.The availability of a specific antibody against native P-LAP permits the rapid purification and the preliminary immunoassay of the enzyme. Establishment of simple purification and assay methods for the native, membrane bound form of P-LAP pave the way to elucidating the roles and processing systems of this enzyme.

An aminopeptidase activity from porcine kidney that hydrolyzes oxytocin and vasopressin: purification and partial characterization

An aminopeptidase from porcine kidney, hydrolyzing oxytocin and vasopressin in vitro, was purified by chromatography on hydroxyapatite, DEAE-cellulose and nickel ion chelate gel and gel filtration on Sephadex G-100. The enzyme appeared to be a high molecular mass ( M r 105 000) monomeric protein. It was sensitive to inhibition by metal chelator, o-phenanthroline. Cobalt ion and sulfhydryl activator, 2-mercaptoethanol, had activating effects, while p-chloromercuribenzoate, amino acids with large hydrophobic side chains, l-cystine and aminopeptidase inhibitors, bestatin and amastatin, had inhibitory effects on the enzyme activity. The enzyme hydrolyzed several aminoacyl p-nitroanilides, and had the highest specificity against S- benzyl- l-cysteine p-nitroanilide. The properties of the enzyme were distinct from those of well-characterized leucyl aminopeptidase (EC 3.4.11.1), membrane alanyl aminopeptidase (EC 3.4.11.2) and primate placental cystinyl aminopeptidase (EC 3.4.11.3).

Evaluation of enzyme inhibitors of cystinyl aminopeptidase and application to the measurement of immunoreactive atrial natriuretic peptide in human pregnancy

Evaluation of enzyme inhibitors of cystinyl aminopeptidase and application to the measurement of immunoreactive atrial natriuretic peptide in human pregnancy