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The activities of alanine aminopeptidase, leucine aminopeptidase, proline dipeptidase and prolyl dipeptidase in the mucosa of the small intestine. Investigations on normal children and patients with the malabsorption syndrome.

Alanine aminopeptidase (EC 3.4.11.2), leucine aminopeptidase (EC 3.4.11.1), proline dipeptidase (EC 3.4.13.9), and prolyl dipeptidase (EC 3.4.13.8) have been investigated in small intestinal mucosa homogenates of normal children and children suffering from different degrees of villous damage. The activities of proline dipeptidase and prolyl dipeptidase could be shown to be significantly decreased in cases of subtotal and total villous atrophy, whereas the activities of alanine aminopeptidase and leucine aminopeptidase were not influenced. The results are discussed in view of the subcellular distribution of these enzymes.

Aminopeptidases↗

Purification, preliminary characterization, and immunological comparison of hog lens leucine aminopeptidase (EC 3.4.11.1) with hog kidney and beef lens aminopeptidases.

Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508-513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53-84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.

Animals↗

A new colorimetric method for the determination of serum enzyme, gamma-glutamyl transpeptidase, cystine aminopeptidase, and leucine aminopeptidase.

A new colorimetric method for the serum enzyme assay was developed, using the color reaction of a ferrous salt complex and a primary aromatic amine. For this method new substrates containing primary aromatic amines were synthesized. The aromatic amine liberated by the enzyme reaction was measured by its color reaction with pentacyanoammineferroate treated with hydrogen peroxide. The determination was made at a wavelength of around 700 nm, at which the assay is not affected by colored substances in serum. Therefore a blank assay was not necessary. This method was applied to the assay of serum gamma-glutamyl transpeptidase, cystine aminopeptidase, and leucine aminopeptidase. The results obtained were in good agreement with those of the p-nitroanilide method also used to assay each enzyme. This new method can be used on a Technicon autoanalyzer system.

Acyltransferases↗

Serum cystine aminopeptidase and leucine aminopeptidase activity in women with benign and malignant uterine and ovarian tumors.

The serum enzymatic activities of cystine aminopeptidase and leucine aminopeptidase were measured in a group of 113 patients of whom 90 had benign uterine or ovarian tumors, and 23 had cancer of the endometrium or ovary. Thirty healthy nonpregnant women and 260 women at different stages of normal pregnancy served as control groups. The presence of pregnancy-specific enzymes in women with uterine or ovarian tumors showed once again that similar processes occur during pregnancy and malignancy. When ovarian or uterine malignancy is suspected on clinical examination, determination of the activities of these enzymes in serum may be of diagnostic value.

Adolescent↗

Downstream change in leucine aminopeptidase activity and leucine assimilation by epilithic microbiota along the River Swale, northern England.

Parallel determinations of epilithic extracellular leucine aminopeptidase activity and leucine assimilation were made at five sites along 112 km of the River Swale and also in two tributaries, the River Wiske and Cod Beck. Epilithic leucine aminopeptidase activity along the Swale increased with distance downstream; this increase was gradual, rather than stepwise in response to specific sewage-works outfalls. Epilithic leucine assimilation, in contrast, did not consistently increase along the river. Epilithic leucine aminopeptidase activity and leucine assimilation were both potentially controlled by epilithic microbial variables (bacterial abundance and chlorophyll a) while leucine aminopeptidase activity was also strongly related to water-quality variables, especially temperature, pH and conductivity. Epilithic leucine aminopeptidase activity and leucine assimilation were coupled, but the magnitude of aminopeptidase activity was always substantially greater than that of leucine assimilation. Arguments are presented, however, which suggest that this did not necessarily indicate the constant availability of excess leucine, and by inference amino-acid nitrogen, to epilithic bacteria. Values of epilithic leucine aminopeptidase activity and leucine assimilation, expressed relative to rates in overlying water, suggested that most activity and assimilation was epilithic rather than planktonic, although the planktonic contribution was proportionately greater at the deeper, more downstream, sites. In the tributaries, River Wiske and Cod Beck, values of epilithic leucine aminopeptidase activity and epilithic microbial abundance, as well as those of many water-quality variables, resembled values in the middle and lower Swale. Thus, these tributaries were essentially lowland, enriched watercourses being very different from the headstreams of the main river.

Animals↗

Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells.

We recently identified two novel aminopeptidases, placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP). Enzymatically, P-LAP degrades oxytocin, vasopressin, and angiotensin III, while A-LAP degrades angiotensin II and kallidin. In this study we investigated the expression and localization of P-LAP and A-LAP in human trophoblastic cells in the normal placenta (n = 26), gestational choriocarcinoma (n = 8), and placental site trophoblastic tumor (n = 3). On immunoblot analysis both P-LAP and A-LAP proteins were detected in normal placenta and five choriocarcinoma tissues, as well as in two choriocarcinoma cell lines. Immunohistochemical staining of normal placental tissues demonstrated that P-LAP was not only localized in villous syncytiotrophoblasts but also highly expressed in extravillous trophoblasts (EVTs) invading the decidua or maternal spiral arteries. The expression level of P-LAP on these invasive EVTs reached a maximum during the late first to second trimesters of pregnancy, and it decreased in the third trimester. Similarly, A-LAP was strongly expressed in EVTs invading the decidua or spiral arteries in the second trimester of pregnancy, while it was weakly or moderately expressed in villous cytotrophoblasts or EVTs located in the cell columns. These two aminopeptidases were more strongly expressed in all eight choriocarcinomas and three placental site trophoblastic tumors and mainly localized to the intermediate-type trophoblastic tumor cells invading the uterine myometrium or stromal vessels. In summary P-LAP and A-LAP were predominantly expressed in the invasive phenotype of EVTs during placentation, as well as in the invasive tumor cells of trophoblastic neoplasms. These results suggest the involvement of these aminopeptidases in invasiveness of both normal and malignant intermediate-type trophoblasts possibly through degradation of specific peptide substrates.

Adipocytes↗

Epilithic and planktonic leucine aminopeptidase activity and leucine assimilation along the River Tweed, Scottish Borders.

The microbial organic-nitrogen transformation variables, extracellular leucine aminopeptidase activity, and leucine assimilation, were determined at stone surfaces and in river water at six sites along 138 km of the River Tweed and also in a major tributary, the River Teviot. Sampling was on 3 days, representative of spring, summer and autumn. The variables, except for epilithic leucine assimilation, all showed significant downstream increase on at least 2 of the sampling days. Their values tended to be lowest in spring (early April). Aminopeptidase activity, leucine assimilation, microbial abundance, and pattern of downstream increase in the Tweed were shown to be broadly similar to in the River Swale, northern England. Microbial organic-nitrogen transformations and abundance in the Teviot tended to equal or exceed those in the lower Tweed, probably because of enrichment of the tributary by sewage-works discharges.

Animals↗

Spin-labeling studies of urea-treated leucine aminopeptidase.

1. Leucine aminopeptidase (EC 3-4-11-1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-1-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B approximately 4:1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) Mn2+ complexed with the protein is partly released producting hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B approximately 4:1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.

Animals↗

Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.

Animals↗

[Influence of glutathione on the catalytic properties of leucine aminopeptidase].

1. Leucine aminopeptidase does not catalyze the hydrolysis of glutathione. 2. Glutathione inhibits the hydrolysis of the substrates leucine hydrazide and leucine-p-nitroanilide by leucine aminopeptidase. 3. By means of kinetic experiments the type of the inhibition has been determined as noncompetitive. The inhibition constant Ki for the Mg2+-activated enzyme is five times higher than for the non-activated enzyme. 4. The degree of inhibition caused by glutathione depends on the pH value indicating a competition between glutathione and OH- ions. Mg2+-activated enzyme is invariably inhibited in the investigated pH range of 7.2 to 9.8. 5. A preincubation of the enzyme with glutathione changes the degree of activity enhancement by metal ions.

Animals↗

[Comparison between leucyl aminopeptidase and pseudo leucine aminopeptidase activities in sera].

The highest activities of leucyl aminopeptidase(LAP, cytosol aminopeptidase, EC 3.4.11.1) in sera have been found in patients with acute hepatitis(Kanno et al., Am J Clin Path, 82: 700-705, 1984). I observed inpatients with very high activities of LAP and alcohol dehydrogenase(AD) in sera. However, only slight elevations of serum pseudo leucine aminopeptidase(PLA), that is, membrane alanyl aminopeptidase(MAA, microsomal aminopeptidase, EC 3.4.11.2) activities for hydrolysis of leucyl-4-nitroanilide were observed in these patients. They were patients in critical care unit with ischemia caused by a cardiopulmonary arrest, multiple trauma, acute myocardial infarction or operation. Therefore, we should measure LAP activities in sera rather than PLA(MAA) activities in these patients.

Acute Disease↗

Effects of progesterone and estradiol on aminopeptidase A (AAP) and leucine aminopeptidase (LAP) activities in the pregnancy serum and placenta of the rat.

We investigated the effects of estradiol and progesterone on placental aminopeptidase A and leucine aminopeptidase activities in pregnant rats. Estradiol (0.5, 1.0 and 2 mg/day) plus progesterone (5, 10 and 20 mg/day) was given for five days starting from the 16th day. Changes in both enzyme activities in the serum, placental cytosol and microsome caused by the steroids were analyzed. Aminopeptidase A activities in all three compartments and leucine aminopeptidase activities in the serum and cytosol were stimulated by estradiol (1.0 mg/day) plus progesterone (10 mg/day). Since we previously found that the placental aminopeptidases degrade peptide hormones, our present study suggests that steroids effect the metabolism of peptide hormones of fetal and/or maternal origin.

Aminopeptidases↗

THE DIAGNOSTIC VALUE OF SERUM LEUCINE AMINOPEPTIDASE.

Serum leucine aminopeptidase determination was found to be a useful screening procedure for hepatobiliary disease in jaundiced and unjaundiced patients. Values under 1,000 units are of no help in the differential diagnosis of jaundice but values above 1,000 units are highly indicative of biliary obstruction. The differentiation of intra- from extrahepatic obstruction as well as of malignant from benign jaundice cannot generally be established by this single test.

Adolescent↗