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At least 1,405 records · Page 78Linked to original sources
PubMed1977

The effect of various drugs on maternal serum cystine aminopeptidase.

Serial estimations (87) of serum cystine aminopeptidase (CAP) in pregnant women (18) using diphenyl hydantoin, phenobarbital or diazepam revealed that the enzyme levels were below the average for normals in 89% of patients. The initially low CAP levels in women on diazepam were improved within 24 h after betamethasone administration. Subsequent studies in vitro confirmed that diphenyl hydantoin, phenobarbital, diazepam and acetyl salicylate markedly inhibited CAP levels in sera of women (72) with clinically uncomplicated pregnancy. No inhibition of CAP by any of the drugs tested was observed in sera from nonpregnant women (8). However, nonpregnant women receiving medroxyprogesterone acetate injections showed CAP inhibition by various drugs, suggesting some changes in the enzyme moiety under the influence of the hormone.

Adolescent↗
PubMed1996

Microinfusion of aminopeptidase M into the paraventricular nucleus of the hypothalamus in normotensive and hypertensive rats.

Normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) received aminopeptidase M (AmM) delivered into the paraventricular nucleus of the hypothalamus (PVN). Resulting changes in blood pressure were recorded in both anesthetized and alert animals. The findings indicate significant dose-determined decreases in blood pressure in members of both strains with SHR more responsive than WKY rats. The respective drops in blood pressure for members within each strain were equivalent for the anesthetized and alert conditions. Pretreatment with the specific angiotensin receptor antagonist, sarthran, [Sar1, Thr2] Angll, into the PVN greatly diminished these responses, suggesting the involvement of the brain angiotensin system. Additionally, a sympathetic nervous system blocker, hexamethonium, and the arginine vasopressin antagonist, Pmp1, O-Me-Tyr2-[Arg] vasopressin, were peripherally administered to assess the potential contributions of these systems to cardiovascular regulation by the brain angiotensin system. The use of these blockers, individually and combined, attenuated responsiveness to infusion of AmM into the PVN. We conclude that AmM can act as a hypotensive agent in both SHR and WKY rats, and that this decrease in blood pressure is at least partially mediated via the brain angiotensin system although other systems may play a role.

Aminopeptidases↗
PubMed2006

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors.

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.

Aminopeptidases↗
PubMed2005

Metal mediated inhibition of methionine aminopeptidase by quinolinyl sulfonamides.

Quinolinyl sulfonamides, such as N-(quinolin-8-yl)methanesulfonamide (10) and N-(5-chloroquinolin-8-yl)methanesulfonamide (11), were identified as potent methionine aminopeptidase (MetAP) inhibitors by high throughput screening of a diverse chemical library of small organic compounds. They showed different inhibitory potencies on Co(II)-, Ni(II)-, Fe(II)-, Mn(II)-, and Zn(II)-forms of Escherichia coli MetAP, and their inhibition is dependent on metal concentration. X-ray structures of E. coli MetAP complexed with 10 revealed that the inhibitor forms a metal complex with the residue H79 at the enzyme active site; the complex is further stabilized by an extended H-bond and metal interaction network. Analysis of the inhibition of MetAP by these inhibitors indicates that this is a typical mechanism of inhibition for many non-peptidic MetAP inhibitors and emphasizes the importance of defining in vitro conditions for identifying and evaluating MetAP inhibitors that will be capable of giving information relevant to the in vivo situation.

Aminopeptidases↗
PubMed2003

Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP).

Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S. salivarius map gene, which codes for MetAP. S. salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6. The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr. The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides. It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+). S. salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C. The S. salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion. A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.

Amino Acid Sequence↗
PubMed2004

Peptidyl hydroxamic acids as methionine aminopeptidase inhibitors.

A new class of methionine aminopeptidase (MetAP) inhibitors, which contain an internal hydroxamate (N-acyl-N-alkylhydroxylamine) core as the metal-chelating group, has been designed, synthesized, and tested. The compounds exhibited reversible, competitive inhibition against Escherichia coli MetAP as well as human MetAP-1 and MetAP-2. The most potent inhibitor had a K(i) value of 2.5 microM and >20-fold selectivity toward E. coli MAP.

Aminopeptidases↗
PubMed2005

Metalloform-selective inhibition: synthesis and structure-activity analysis of Mn(II)-form-selective inhibitors of Escherichia coli methionine aminopeptidase.

Methionine aminopeptidase (MetAP) is a promising target for development of novel antibacterial, antifungal and anticancer agents. However, its physiologically relevant metal ion remains to be defined, and its inhibitors need to inhibit the in vivo metalloform. Based on the Mn(II)-form-selective inhibitors discovered by high throughput screening as leads, a series of analogs of 5-phenylfuran-2-carboxylic acid was prepared and subsequently evaluated on Co(II)-, Mn(II)-, Ni(II)-, and Fe(II)-forms of Escherichia coli MetAP, in order to define the structural elements responsible for their inhibitory potency and metalloform selectivity. Various substitutions on the phenyl ring changed their potency on the Mn(II)-form but not their metalloform selectivity. We conclude that the preferential coordination of the carboxyl group to Mn(II) ions is the major determinant for their superb selectivity toward the Mn(II)-form. Changing the carboxylate to hydroxamate alters its ability to bind and discriminate different metal ions, and the hydroxamate derivative becomes non-selective among the metalloforms tested.

Aminopeptidases↗
PubMed2004

Chronic ethanol intake modifies renin-angiotensin system-regulating aminopeptidase activities in mouse cerebellum.

In developing cerebellum, where critical periods of vulnerability have been established for several basic substances, it has been extensively studied the wide array of abnormalities induced by exposure to ethanol (EtOH). However, little is known about the effects of EtOH consumption on cerebellar functions in adult individuals. Several studies show participation in cognitive activities to be concentrated in the lateral cerebellum (hemispheres), whereas basic motor functions such as balance and coordination are represented in the medial parts of the cerebellum (vermis and paravermis). In addition to the circulating renin angiotensin system (RAS), a local system has been postulated in brain. The effector peptides of the RAS are formed via the activity of several aminopeptidases (AP). The present work analyses the effect of chronic EtOH intake on the RAS-regulating AP activities in the soluble and membrane-bound fractions of two cerebellar locations: the hemispheres and the vermis. We hypothesize that cerebellar RAS is involved in basic motor functions rather than in cognitive activities.

Aminopeptidases↗
PubMed1980

Early primary diagnosis of ovarian cancer and detection of recurrence by serum cystine aminopeptidase assay.

The aim of this study was to correlate the relationship between serum cystine aminopeptidase (CAP) activity in normal patients and that in patients with primary ovarian cancer of recurrence. Sera obtained from 63 patients and evaluated for CAP activity by the method of Babuna and Yenen were grouped as follows: in 20 normal, nonpregnant patients, the mean activity was 25; in 14 patients with fibromas of the uterus, it was 38; in two patients with ovarian sarcoma, the activities were "normal" (24 to 27); and in 27 patients with primary ovarian adenocarcinoma or recurrence, the activity corresponded to the size of the tumors. Assay of serum CAP activity is a simple procedure which can be performed in any laboratory. It is a quick method for qualitative, and even quantitative, assessment of an ovarian adenocarcinoma in the nonpregnant patient. It may prove to be especially valuable in the follow-up of patients with ovarian cancer.

Adenocarcinoma↗
PubMed2002

Protein kinase C-zeta phosphorylates insulin-responsive aminopeptidase in vitro at Ser-80 and Ser-91.

Insulin-responsive aminopeptidase (IRAP) colocalizes with glucose transporter type 4 (GLUT4) in adipocytes and is recruited to the plasma membrane in response to insulin. Microinjection of peptides corresponding to the IRAP cytoplasmic domain sequences causes GLUT4 recruitment in adipocytes. Inhibitors of protein kinase C-zeta (PKC-zeta) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-zeta may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the (32)P incorporation into IRAP catalyzed by PKC-zeta in insulin-stimulated cells. There was a small but significant (32)P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-zeta pseudosubstrate, suggesting that PKC-zeta may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-zeta also catalyzed the incorporation of (32)P not only into IRAP in GLUT4 vesicles isolated from rat adipocytes but also into the IRAP cytoplasmic domain inserts in glutathione S-transferase-fusion proteins, demonstrating direct IRAP phosphorylation by PKC-zeta. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the (32)P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the (32)P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP(76-138) and the (32)P labeling at Ser-80 accounted for 80-90% of the total IRAP labeling, suggesting that Ser-80 is the major phosphorylation site in intact IRAP. These findings are consistent with the possibility that the IRAP cytoplasmic domain phosphorylation by PKC-zeta plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.

Adipocytes↗
PubMed2003

Specificity for inhibitors of metal-substituted methionine aminopeptidase.

Methionine aminopeptidases (MetAPs) have been studied in vitro as Co(II) enzymes, but their in vivo metal remains to be defined. While activation of Escherichia coli MetAP (EcMetAP1) by Co(II), Mn(II), and Zn(II) was detectable by a colorimetric Met-S-Gly-Phe assay, significant activation by Ni(II) was shown in a fluorescence Met-AMC assay, in addition to Co(II) and Mn(II) activation. When tested on the metal-substituted EcMetAP1s, a few inhibitors that we obtained recently from a random screening on Co-EcMetAP1 either became much weak or lost activity on Mn- or Zn-EcMetAP1, although they kept inhibitory activity on Ni-EcMetAP1. A couple of peptidic inhibitors and the methionine mimetic (3R)-amino-(2S)-hydroxyheptanoic acid (AHHpA, 6) maintained moderate activities on Co-, Mn-, Zn-, and Ni-EcMetAP1s. Our results clearly demonstrate that the metal-substitution has changed the enzyme specificity for substrates and inhibitors. Therapeutic applications call for inhibitors specific for MetAP with a physiologically relevant metal at its active site.

Aminopeptidases↗
PubMed1975

A rapid and specific method for the determination of oxytocinase (cystine aminopeptidase) activity using continuous flow analysis and S-benzyl-L-cysteine-p-nitroanilide as substrate.

1. An automated method is described for the assay of pregnancy serum cystine aminopeptidase (Oxytocinase, E.C. 3.4.1.10) using continuous flow analysis. S-Genzyl-L-cysteine-p-nitroanilide, the most highly specific substrate so far available for oxytocinase is employed in this assay. Comparison with the manual method from which it has been developed, gave a correlation coefficient of 0.98. Reaction of oxytocinase on the substrate liberated p-nitroaniline, the absorption of which is measured at 410nm. 2. Blank values are eliminated by the incorporation of a dialyzer in the manifold design and thereby effectively increasing the rate of assay sample to 40 per hour. The lag phase (a) of the continuous flow analysis is 0.17 min while the exponential phase (b) is 0.31 min. Using a 2:1 sample to wash ration the carry-over is 3% at a 95% of steady states.

Aminopeptidases↗
PubMed1983

Predictive value analysis of measurements of human chorionic gonadotropin, pregnancy specific beta 1-glycoprotein, placental lactogen, and cystine aminopeptidase for the diagnosis of ectopic pregnancy.

The diagnostic utility of serum and urinary human chorionic gonadotropin (hCG) measurements and serum measurements of pregnancy-specific beta 1-glycoprotein (PSBG), placental lactogen, and cystine aminopeptidase (CAP) was prospectively studied in 51 consecutive patients with suspected ectopic pregnancy who underwent laparoscopy or laparotomy. CAP was not detected in the sera of any patient with ectopic pregnancy. False-positive results in each assay were found in patients with intrauterine pregnancy or missed abortion. The overall efficiency of the tests were: urine hCG slide test, 47%; urine hCG tube test, 80%; serum hCG, 86%; serum PSBG, 82%; and human placental lactogen, 60%. Although measurement of serum hCG by radioimmunoassay is the most accurate biochemical test in predicting the correct diagnosis in patients with suspected ectopic pregnancy, the relatively high efficiency of the urinary hCG tube test, coupled with its ease of performance, low cost, and rapid turnaround time, makes it the most practical screening test.

Aminopeptidases↗
PubMed2003

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors.

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.

Amino Acid Sequence↗
PubMed1998

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide.

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.

Aminopeptidases↗
PubMed2000

Structure and function of the methionine aminopeptidases.

The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.

Aminopeptidases↗
PubMed2002

Effects of age, hypertension and HRT on serum aminopeptidase A activity.

OBJECTIVES: It is conceivable that aminopeptidase A (APA)/angiotensinase is involved in the pathogenesis of hypertension. The aim of this study was to evaluate the influences of age, hypertension and hormone replacement therapy (HRT) on serum APA activity in middle-aged and elderly women. METHODS: Blood samples were collected from 117 women aged 40-69, 48 normotensive healthy women not receiving HRT, 57 normotensive women receiving HRT, and 12 hypertensive women (blood pressure >140/90 mmHg) with no medication. Serum APA activity was measured using alpha-glutamyl-p-nitroanilide as substrate spectrophotometrically. RESULTS: Serum APA activity increased along with age between 40 and 69 in healthy women not taking HRT (r=0.351, P<0.05). Hypertensive women had higher serum APA activity than age-matched normotensive women (25.4+/-4.2 versus 22.4+/-3.4 microM/min; P<0.05) Compared with non-users of HRT, APA activity was elevated in women receiving HRT (23.9+/-3.8 versus 20.4+/-3.2 microM/min; P<0.05). CONCLUSIONS: APA activity had a positive correlation with age in healthy women. Furthermore, hypertension and HRT up-regulated the serum APA activity significantly. The measurements of serum APA would be of value to elucidate the physiological and clinical roles of APA, although further studies are required.

Adult↗
PubMed1998

N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families.

Removal of the initiator methionine and/or acetylation of the alpha-amino group are among the earliest possible chemical modifications that occur during protein synthesis in eukaryotes. These events are catalyzed by methionine aminopeptidase and N alpha-acetyltransferase, respectively. Recent advances in the isolation and characterization of these enzymes indicate that they exist as isoforms that vary in cellular location, function, and evolutionary origins.

Acetyltransferases↗