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PubMed1993

Differentiation and identification of the two catalytic metal binding sites in bovine lens leucine aminopeptidase by x-ray crystallography.

The tightly binding and readily exchanging metal binding sites in the active site of bovine lens leucine aminopeptidase (blLAP; EC 3.4.11.1) have been differentiated and identified by x-ray crystallography. In native blLAP,Zn2+ occupies both binding sites. In solution, site 1 readily exchanges Zn2+ for other divalent cations, including Mg2+. The Zn2+ in site 2 is unavailable for metal exchange under conditions which allow exchange at site 1. The Zn2+/Mg2+ metal hybrid of blLAP (Mg-blLAP) was prepared in solution and crystallized. X-ray diffraction data to 2.9-A resolution were collected at -150 degrees C from single crystals of Mg-blLAP and native blLAP. Comparisons of omit maps calculated from the Mg-blLAP data with analogous maps calculated from the native blLAP data show electron density in one of the metal binding sites in Mg-blLAP which is much weaker than the electron density in the other binding site. Since there are fewer electrons associated with Mg2+ than with Zn2+, the difference in electron density between the two metal binding sites is consistent with occupancy of the weaker electron density site by Mg2+ and identifies this metal binding site as site 1, defined as the readily exchanging site. The present identification of the metal binding sites reverses the previous presumptive assignment of the metal binding sites which was based on the structure of native blLAP [Burley, S. K., David, P. R., Sweet, R. M., Taylor, A. & Lipscomb, W. N. (1992) J. Mol. Biol. 224, 113-140]. According to the residue-numbering convention of native blLAP, the new assignment of the metal binding sites identifies the readily exchanging site 1 with Zn-488, which is within interaction distance of one side-chain carboxylate oxygen from each of Asp-255, Asp-332, and Glu-334 and the main-chain carbonyl oxygen of Asp-332. The more tightly binding site 2 is identified with Zn-489, which is within interaction distance of one side-chain carboxylate oxygen from each of Asp-255, Asp-273, and Glu-334 and the side-chain amine nitrogen of Lys-250.

Amino Acid Sequence↗

PubMed1999

Serum leucine aminopeptidase as an activity indicator in systemic lupus erythematosus: a study of 46 consecutive cases.

OBJECTIVE: To determine whether elevations in serum leucine aminopeptidase (LAP) levels reflected the underlying evolution of active disease in systemic lupus erythematosus (SLE). METHODS: We studied serum LAP levels, other laboratory indicators, and SLE Disease Activity Index (SLEDAI) scores, in 46 consecutive patients with SLE admitted to Tokyo Metropolitan Komagome Hospital. LAP levels in 46 patients with rheumatoid arthritis were also measured. RESULTS: Thirty-three SLE patients had elevated LAP levels. LAP levels correlated positively with levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transpeptidase, and negatively with the total serum haemolytic complement and leucocyte, neutrophil and lymphocyte counts, but showed no correlation with alkaline phosphatase, gamma-globulin, beta2-microglobulin or C-reactive protein levels, or platelet count. The SLEDAI score correlated positively with LAP levels. The LAP level in patients with rheumatoid arthritis was near normal. CONCLUSION: The serum LAP level may be a potential activity indicator for SLE.

Adult↗

PubMed2001

Leucine aminopeptidase as a target for inhibitor design.

In this review we focus on the most effective and the most promising inhibitors of leucine aminopeptidase. Their binding modes to the enzyme, the attempt to explain the origin of the inhibitory activity, as well as the structure-activity relationship for some of these compounds are discussed. Besides, the structural and electronic requirements of the enzyme active site and the binding pockets, together with the specificity towards the ligands, based on the structural and kinetic data, are presented.

Amino Acids↗

PubMed1976

[Activation of leucine aminopeptidase under hydrogen bond cleaving conditions].

Cleavage of hydrogen bonds by urea, guanidinium chloride or elevated temperatures causes a reversible activation of leucine aminopeptidase. The activation is similar to that caused by Mg2+ ions. This means that preincubation is required and that a 10-fold or more activated enzyme is inhibited by 50 mM cyanide to 20 per cent while a C1-ion-activated enzyme like the nonactivated enzyme is inhibited to 90 per cent. Blockage of the free SH-groups reduces the response time of the activation. The free SH-groups are involved in an essential intermediate step of the activation.

Enzyme Activation↗

PubMed1998

Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase.

Most antigenic peptides presented on major histocompatibility complex class I molecules are generated during protein breakdown by proteasomes, whose specificity is altered by interferon-gamma (IFN-gamma). When extended versions of the ovalbumin-derived epitope SIINFEKL are expressed in vivo, the correct C terminus is generated by proteasomal cleavage, but distinct cytosolic protease(s) generate its N terminus. To identify the other protease(s) involved in antigen processing, we incubated soluble extracts of HeLa cells with the 11-mer QLESIINFEKL, which in vivo is processed to the antigenic 8-mer (SIINFEKL) by a proteasome-independent pathway. This 11-mer was converted to the 9-mer by sequential removal of the N-terminal residues, but surprisingly the extract showed little or no endopeptidase or carboxypeptidase activity against this precursor. After treatment of cells with IFN-gamma, this N-terminal trimming was severalfold faster and proceeded to the antigenic 8-mer. The IFN-treated cells also showed greater aminopeptidase activity against many model fluorogenic substrates. Upon extract fractionation, three bestatin-sensitive aminopeptidase peaks were detected. One was induced by IFN-gamma and was identified immunologically as leucine aminopeptidase (LAP). Purified LAP, like the extracts of IFN-gamma-treated cells, processed the 11-mer peptide to SIINFEKL. Thus, IFN-gamma not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can stimulate formation of their N termini by inducing LAP. This enzyme appears to catalyze the trimming of the N terminus of this and presumably other proteasome-derived precursors. Thus, susceptibility to LAP may be an important influence on the generation on immunodominant epitopes.

Antigens↗

PubMed1992

Chicory seed lot variety identification by leucine-aminopeptidase and esterase zymogram analysis.

A method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility-density scheme. This quick, easy and low-cost method can be a useful tool for chicory breeders.

Electrophoresis, Polyacrylamide Gel↗

PubMed1977

Influence of L-thyroxine upon enzymatic activity in the renal tubular epithelium of the rat under normal conditions and in mercury-induced lesions. I. Histochemical studies of alkaline phosphatase, acid phosphatase, adenosine- tri-phosphatase and leucine-aminopeptidase.

HgC12-induced renal tubular lesions in the rat present histochemically with a transitory decrease of alkaline phosphatase, adenosinetriphosphatase (ATPase), and leucine-aminopeptidase activity. The toxic alterations of enzyme activity were more pronounced in the pars recta of the proximal tubule and in the loop of Henle, as compared with the tubulus contortus I. L-thyroxine treatment leads to an accelerated reversal of that enzymatic defect, followinga characteristic pattern, and to a differentiating increase of acid phosphatase and ATPase activity in certain parts of the normal renal tubule. The observations are discussed with reference to the specific mode of action of sublimate and l-thyroxine upon the tubular enzymes and to the well-known metabolic and functional influences of thyroid hormone on the kidney.

Acid Phosphatase↗

PubMed2001

The induction of tomato leucine aminopeptidase genes (LapA) after Pseudomonas syringae pv. tomato infection is primarily a wound response triggered by coronatine.

Tomato plants constitutively express a neutral leucine aminopeptidase (LAP-N) and an acidic LAP (LAP-A) during floral development and in leaves in response to insect infestation, wounding, and Pseudomonas syringae pv. tomato infection. To assess the physiological roles of LAP-A, a LapA-antisense construct (35S:asLapA1) was introduced into tomato. The 35S:asLapA1 plants had greatly reduced or showed undetectable levels of LAP-A and LAP-N proteins in healthy and wounded leaves and during floral development. Despite the loss of these aminopeptidases, no global changes in protein profiles were noted. The 35S:asLapA1 plants also exhibited no significant alteration in floral development and did not impact the growth and development of Manduca sexta and P. syringae pv. tomato growth rates during compatible or incompatible infections. To investigate the mechanism underlying the strong induction of LapA upon P. syringae pv. tomato infection, LapA expression was monitored after infection with coronatine-producing and -deficient P. syringae pv. tomato strains. LapA RNA and activity were detected only with the coronatine-producing P. syringae pv. tomato strain. Coronatine treatment of excised shoots caused increases in RNAs for jasmonic acid (JA)-regulated wound-response genes (LapA and pin2) but did not influence expression of a JA-regulated pathogenesis-related protein gene (PR-1). These results indicated that coronatine mimicked the wound response but was insufficient to activate JA-regulated PR genes.

Amino Acids↗

PubMed1996

Urinary leucine aminopeptidase is a more sensitive indicator of early renal damage in non-insulin-dependent diabetics than microalbuminuria.

We measured urinary activity of leucine aminopeptidase (EC 3.4.11.2) and creatinine concentrations (Cr, in mmol) in samples of second morning urine from 25 healthy subjects and 59 non-insulin-dependent diabetic (NIDD) subjects. If NIDD subjects are grouped according to their Alb/Cr ratio into normoalbuminuria (group A, Alb/Cr < 2.8 mg/mmol), microalbuminuria (group B, Alb/Cr 2.8-26.8 mg/mmol), and macroalbuminuria (group C, Alb/Cr > 26.8 mg/mmol), LAP/Cr ratios in all three groups exceeded those for healthy age-matched controls. Moreover, this ratio was higher in group B than in group A. The value for LAP/Cr was clearly abnormal (i.e., exceeded the upper limit of normal, log normal + 2 SD, found in healthy subjects) in 44% of group A. In the first 10-year period Of NIDD, prevalance of abnormal LAP/Cr ratio was 61.3%, whereas that of microalbuminuria was 35.5%. We have also found a LAP/Cr ratio abnormality of 91% in group B. Evidently, LAP/Cr may be increased early in NIDD subjects and be a more sensitive predictor of incipient nephropathy than microalbuminuria.

Adult↗

PubMed1982

Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins.

Rabbit antisera to bovine lens leucine aminopeptidase (LAP) have been prepared. These LAP-specific antisera cross-react with a component, LAP, in human lens homogenates. Bovine and human lens LAP are similar but not identical. Immunodiffusion tests show that LAP is present in a vast majority if not all cataractous and normal human lens homogenates. Results from immunoelectrophoresis indicate that LAP is found in these homogenates as several metazymes as well as in an albuminoid--and possibly membrane-associated form. In contrast to many activity-based studies which imply that very little LAP is present in human lenses, micro-complement fixation tests indicate that the concentration of LAP in aged human lenses is similar to that found in calf lenses. Taken together, these data indicate that LAP undergoes age-related changes. These alterations of enzyme (or environment) result in an enzyme of markedly reduced activity, hence, the discrepancy between amount of LAP and the amount of enzymatic activity in aged human lenses. Active LAP is shown to enhance the rate of hydrolysis of alpha-2 and alpha-B crystallins but not alpha-A2, beta H, beta L or gamma crystallins. The ramifications of LAP inactivation with respect to cataractogenesis are discussed.

Adult↗

PubMed1981

Urine and serum leucine aminopeptidase, N-acetyl-beta-glucosaminidase and gamma-glutamyl transpeptidase activities in diabetics with and without nephropathy.

Urinary and serum activities of leucine aminopeptidase (LAP), N-acetyl-beta-glucosaminidase (ABG) and gamma-glutamyl transpeptidase (GGTP) were determined in four groups: 1) control subjects with normal oral glucose tolerance test and normal renal function; 2) latent diabetics with elevated oral glucose tolerance test and diabetic parents; 3) overt diabetics; 4) diabetics with slight nephropathy, exemplified by elevated serum creatinine levels and decreased creatinine clearance. No clear differences were found in the urinary GGTP and serum enzyme activities. Urinary LAP activity was significantly increased in Groups 2, 3 and 4, and urinary ABG activity in Groups 3 and 4. However, since the rise in urinary LAP activity was less consistent than that in ABG, it seems that the appearance of the latter enzyme in urine most likely results from tubular damage and may serve as a sensitive indicator of early diabetic kidney disease.

Acetylglucosaminidase↗

PubMed1993

X-ray crystallographic determination of the structure of bovine lens leucine aminopeptidase complexed with amastatin: formulation of a catalytic mechanism featuring a gem-diolate transition state.

The structure of the complex of bovine lens leucine aminopeptidase (blLAP) with the slow-, tight-binding inhibitor amastatin has been determined by X-ray crystallography. X-ray diffraction data were collected at -150 degrees C from a single blLAP-amastatin crystal which under the data collection conditions was of the space group P6(3)22 with unit cell parameters a = 130.3 A and c = 121.9 A. The structure of the blLAP-amastatin complex was determined by molecular replacement, using the structure of native blLAP as the starting model. Refinement of the blLAP-amastatin model plus 132 water molecules against data from 10.0- to 2.4-A resolution resulted in a final structure with a crystallographic residual of 0.198. The binding mode of amastatin is similar to that of bestatin, the structure of whose complex with blLAP has previously been determined. Of particular note, the N-terminus-to-C-terminus orientation of the two bound inhibitors is the same. The two N-terminal residues of amastatin and bestatin occupy the same binding sites, which are most likely S1 and S'1. The slow binding of amastatin and bestatin may be partially attributable to a binding mechanism in which the two active site metals are sequentially coordinated by the P1 amino and hydroxyl groups of these inhibitors. A catalytic mechanism for blLAP is proposed based on the binding modes of amastatin and bestatin and plausible binding modes of a dipeptide substrate and its putative gem-diolate transition state which were modeled into the active site of blLAP after the binding mode of amastatin. The proposed catalytic mechanism invokes roles for the catalytic metals in binding and activating the substrate and in stabilizing the transition state. The mechanism also includes roles for Asp-255 as a general base, Arg-336 as an additional electrophilic substrate activator and transition state stabilizer, and Lys-262 as a proton shuttle.

Amino Acid Sequence↗

PubMed2000

Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated. The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E. coli PepA markedly. Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position. P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs.

Animals↗

PubMed2002

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

Amino Acid Sequence↗

PubMed1979

Effect of diets supplemented with amino acids on intestinal sucrase and leucine aminopeptidase activities in rats.

In order to investigate the relationship between dietary amino acids and protein, and activities of intestinal sucrase [EC 3.2.1.26] and leucine aminopeptidase [EC 3.4.11.1, LAPase] in rats, the effect of supplementation of amino acids into a protein-free diet and a low casein diet containing sucrose as the carbohydrate source on these enzyme activities was studied. The segmental weights of the small intestine and its mucosa of rats fed the protein-free diet supplemented with L-methionine or with L-methionine and L-threonine at 0.1 or 0.2% levels were significantly higher than those of rats fed the protein-free diet or one supplemented with L-glutamic acid, but there was no difference in the segmental activities of the sucrase and LAPase among rats fed these diets. On the other hand, the supplementation of methionine or methionine plus threonine to the 5% or 10% casein diet produced remarkable increases in the segmental weights of the small intestine and its mucosa as well as in the segmental activities of the sucrase and LAPase. There was no difference between the segmental sucrase activity of rats fed the 10% casein diet supplemented with 0.2% methionine ad libitum and that of rats fed this diet under restricted feeding conditions, although the segmental LAPase activity was affected by the amount of food consumed.

Amino Acids↗

PubMed1978

Special features of intestinal metaplasia and its relation to early gastric carcinoma in man: observation by a method in which leucine aminopeptidase activity is used.

The degree of intestinal metaplasia and its macroscopic distribution in gastric mucosa were examined by a new method in which leucine aminopeptidase (LAP) activity is used to investigate the relationship between intestinal metaplasia and gastric carcinoma. Since LAP is a specific enzyme of intestinal metaplasia, the area showing positive reaction for this enzyme corresponds strictly with the zone of intestinal metaplasia examined microscopically. Almost all metaplasia was demonstrated by this method. This method was used to examine 40 human gastric carcinomas confined to the mucosa. Gastric mucosa containing differentiated tubular-type carcinoma (60%) was associated with a high degree of metaplasia in comparison with the low degree of metaplasia in poorly differentiated carcinoma (40%). Differentiated tubular-type carcinoma was closely related to intestinal metaplasia. However, all carcinomas arising from the mucosa without intestinal metaplasia (18%) were poorly differentiated. Therefore, gastric carcinomas occurring in Japanese patients are frequently and closely related to intestinal metaplasias.

Alkaline Phosphatase↗

PubMed1979

The LAP test. A new method for identification of seminal stains by a qualitative color reaction of leucine aminopeptidase.

A new simple method for identification of seminal stains is described. It employs a qualitative color reaction based on histochemical technique for demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen. The method herein reported (the LAP test) is quite suitable for medicolegal examination of seminal stains as a preliminary test.

Color↗

PubMed1985

Plasma angiotensin I and serum placental leucine aminopeptidase (P-LAP) in pre-eclampsia.

A study was undertaken on serial measurements of plasma angiotensin I (A-I) and serum placental leucine aminopeptidase (P-LAP) activities in normal and pre-eclamptic pregnancy. There was the significant difference in A-I levels between normal and mild pre-eclamptic pregnancy at weeks 30, 35, 37, between normal and severe pre-eclamptic pregnancy at week 37. There were no differences in serum P-LAP between normal and mild pre-eclamptic pregnancy up to week 33, but thereafter the levels for the mild pre-eclampsia were significantly higher than for the normal pregnancy. The P-LAP activity for the severe pre-eclampsia reached its maximum level at week 31. Around this week, the levels for severe pre-eclampsia were significantly higher than in the normal pregnancy. After week 35, the activities decreased precipitously to week 40; the activities for severe pre-eclampsia in late pregnancy at weeks 39 and 40 were significantly lower than in normal pregnancy. The above data support the idea that P-LAP test is useful for prediction or diagnosis of pre-eclampsia.

Adult↗