The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases.

The suitability of hexazonium-p-rosanilin (HP) in the histochemical demonstration of peptidases was investigated. The detection was carried out in cold mictrotome sections adherent to slides or semipermeable membranes. Alanyl-1-naphthylamide, alanyl-2-naphthylamide, leucyl-2-naphthylamide, leucyl-4-methoxy-2-naphthylamide (all substrates in concentration of 0.4 mg/1 ml of citrate phosphate buffer pH 6.5), gamma-L-glutamyl-1-naphthylamide, gamma-L-glutamyl-2-naphthylamide (both substances in concentration of 0.24 mg/1 ml of acetate buffer pH 6.5) were used as the substrates. Results were compared with those obtained with Fast Blue B and Fast Garnet GBC. In comparison with Fast Blue B and Fast Garnet GBC HP is a faster coupler, furnishes azodyes which are stable, amorphous (even without lipid extractions from sections), more substantive and in the case of 1-naphthylamine almost insoluble in ordinary lipid solvents used for the dehydration and clearing of sections before mounting. The molecular extinction coefficient of azodyes furnished by HP is 1.5X higher for 1-naphthylamine than for 2-naphthylamine. It is higher than that of Fast Garnet GBC, however, lower than that of Fast Blue B. The inhibitory influence of individual diazonium salts on enzyme activity (activities) splitting leucyl-2-naphthylamide amounts to 36% (Fast Garnet GBC), 37% (Fast Blue B), 52% (HP, 0.03 ml/1 ml) and 63% (HP, 0.09 ml/1 ml) at pH 6.5. For gamma-glutamyl-transpeptidase the corresponding values are 50%, 59%, 62% and 67%. The higher inhibitory influence of HP is compensated by the possibility of its using in the technic of semipermeable membranes. HP improves greatly the localization of peptidases in cold microtome sections from which lipids were not extracted. The best results are furnished by 1-naphthylamine dervatives. In the case of 4-methoxy-2-naphthylamine derivatives the localization is very sharp, however, the azodye is less distinct than that of 2-naphthylamine. The localization as obtained with HP in combination with substrates derived of simple naphthylamines is similar or even better than with 4-methoxy-2-naphthylamine derivatives applied with Fast Blue B. Typical examples are shown.     

A fluorescent assay of proteinases in cultured mammalian cells

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.     

Use of peptidyl-4-methoxy-2-naphthylamides to assay plasmin

Several tripeptide amides of 4-methoxy-2-naphthylamide were synthesized and tested as possible substrates for human plasmin. These peptides contained arginine or lysine as the carboxyl terminus. One such amide, benzyloxycarbonyl-Ala-Ala-Lys-4-methoxy-2-naphthylamine (CBZ-Ala-Ala-Lys-MNA), was found to be a better substrate for plasmin than for other mammalian serine proteases. Measuring the release of 4-methoxy-2-naphthylamine fluorometrically or by colorimetry after coupling with fast blue B dye provided convenient assays for as little as 0.05 CTA unit of plasmin. K_m and k_cat values obtained were 0.90 m and 0.7 sec⁻¹, respectively. The assays were simple, sensitive, versatile, and specific.     

Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF)

Summary A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and -glutamyltranspeptidase (GGT) show striking heterogenous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.     

Identification of neuropeptide-degrading enzymes in the pancreas.

Neutral endopeptidase (NEP) and aminopeptidase M (APM) were identified in the pancreas by enzymatic assays and Western blotting. The NEP activity, assessed by the phosphoramidon- and DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was 28.8 pmol/h/micrograms of pancreatic membrane protein and 124 pmol/h/10(6) pancreatic acinar cells. The APM enzymatic activity, assessed by the actinonin- and amastatin-inhibitable degradation of Ala-4-methoxy-2-naphthylamine, was 633 pmol/h/micrograms pancreatic membrane protein and 17.4 nmol/h/10(6) pancreatic acinar cells. Proteins corresponding to NEP (95 kDa) and APM (140 kDa) were identified in membranes by Western blotting. Both NEP and APM on acinar cells may degrade neuropeptides and regulate their effects on exocrine secretion.     

Uptake of exogenous metabolites by virus-transformed cells: changes induced by temperature and pH.

Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-(S)-leucine, inhibited aminopeptidase B and leucine aminopeptidase in a competitive manner and their K_i values were calculated to be 6 × 10^?8 and 2 × 10^?8M, respectively. Among all stereoisomers of bestatin synthesized, those which have a (2S)-configuration in the 3-amino-2-hydroxy-4-phenylbutanoyl moiety showed marked inhibition against aminopeptidase B and leucine aminopeptidase compared with the other isomers which have (2R)-configuration. One of the isomers, [(2S,3S)-3-amino-2-hydroxy-4-phenylbutanoyl]-(R)-leucine, showed somewhat stronger activity against aminopeptidase B than bestatin. Aminopeptidase B appears to be a metallo-exopeptidase. It is proposed that bestatin and its active isomers are effective due to a mechanism other than a chelating action at the active center.     

Localization of leucine aminopeptidase and vitamin B-12 binding protein in rabbit peripheral blood polymorphonuclear leukocytes

Polymorphonuclear leukocytes were isolated from the peripheral blood of rabbits by Ficoll-Hypaque centrifugation followed by dextran sedimentation. The granulocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. Leucine aminopeptidase, when assayed with L-leucine-7-amido-4-methyl-coumarin as substrate, showed a similar distribution to N-acetyl-ß-glucosaminidase and thus is localized to the tertiary granules. There was no leucine aminopeptidase associated with the plasma membrane of these cells. Further experiments with purified plasma membranes and inhibitor studies using diazotized sulphanilic acid further confirmed that leucine aminopeptidase had a purely intracellular localization. Vitamin B-12 binding protein showed a similar localization to alkaline phosphatase indicating that, as in human polymorphonuclear leukocytes, vitamin B-12 binding protein is located to the specific granules.     

Bitter Peptide Isolated from Milk Cultures of Streptococcus cremoris

Certain cultures of Streptococcus cremoris produced a bitter taste that occurred in the whey portion of milk cultures. Whey from a culture which produced bitterness was fractionated on Sephadex. The fraction in which the bitter taste was concentrated was chromatographed successively on paper with butanol-acetic acid-water (5:1:4), and then butanol-2-butanone-water (2:2:1). In each instance, the bitter component was in the most rapidly moving band that gave a positive ninhydrin test. The bitterness was observed to be caused by a peptide containing the following numbers of each amino acid: arginine, 1; glutamic acid, 2; glycine, 2; isoleucine, 2; leucine, 2; phenylalanine, 1; proline, 5; and valine, 4. N-terminal amino acids could be detected by coupling with 2,4-dinitrofluorobenzene or phenylisothiocyanate, or by hydrolysis with leucine aminopeptidase. When treated with carboxypeptidase, only leucine and valine appeared at the C-terminal end, and these were detected simultaneously.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates

Summary In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited y diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methylcoumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.     

Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen.

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.     

Inactivation of phagocytosis-stimulating activity of tuftsin by polymorphonuclear neutrophils: A possible role of leucine aminopeptidase as an ecto-enzyme

The subcellular localization of the tuftsin-inactivating activity was studied using guinea-pig polymorphonuclear neutrophils and the following results were obtained. 1. The tuftsin-inactivating activity was present in the membrane function but not in the cytosol and the granular fractions. 2. Intact neutrophils inactivated tuftsin rapidly. However, when neutrophils were modified chemically by a poorly permeant reagent, diazotized sulfanilic acid, the tuftsin-inactivating activity decreased sifnificantly without any inhibition of marker enzymes of cytosol, microsome, granulesa and mitochondria, suggesting that the tuftsin-inactivating activity is located on the plasma membrane as an ecto-enzyme. 3. When neutrophils were modified by diazotized sulfanilic acid at different concentrations, the tuftsin-inactivating activity of neutrophils was inhibited in proportion to the degree of inhibition of the activity of leucine aminopeptidase, an ecto-enzyme. 4, Hydrolysis of L-leucyl-β-napthylamide, a synthetic substrate of leucine aminopeptidase, was inhibited competitively by tuftsin. 5. Treatmetn of neutrophils with serine protease inhibitors affected neither tuftsin-inactivating nor leucine aminopeptidase activity at all, indicating no involvement of serine proteases, which is said to be located on the cell surface membrane, in the tuftsin-inactivating activity of neutrophils. The possibility was deduced from the above results that leucine aminopeptidase may act as a tuftsin-inactivating enzyme.     

Demonstration of protease isoenzymes by two-dimensional polyacrylamide electrophoresis

A two-dimensional polyacrylamide electrophoresis system is described for the detection of protease isoenzymes using 4-methoxy-2-naphthylamine derivates as substrates. With this technique is possible to detect isoenzymes differing in molecular weight by separation according to size and shape. This is not possible by the use of isoelectric focusing alone.     

Purification and properties of an extracellular leucine aminopeptidase from the Bacillus sp. N2

A halophilic bacterium was isolated from fermented anchovy sauce and identified as Bacillus species. An extracellular leucine aminopeptidase from Bacillus sp. N2 was purified to homogeneity using four successive purification steps. The enzyme has a native molecular mass of about 57 000 Da using FPLC gel filtration analysis and a molecular mass of 58 000 Da using SDS-polyacrylamide gel electrophoresis. This monomeric leucine aminopeptidase showed maximum enzyme activity at pH 9·5. The optimum temperature was 50 °C when L -Leu- p -nitroanilide was the substrate. The leucine aminopeptidase was inactivated by 1,10-phenanthroline, dithiothreitol and sodium dodecyl sulphate. Enzyme activity was increased by addition of Co²⁺. It is likely that Co²⁺ plays an important role in the catalysis or stability of the Bacillus sp. N2 leucine aminopeptidase. Sodium chloride (0–4·5 mol l⁻¹) increased the hydrolytic activity towards L -Leu- p -nitroanilide. The N-terminal amino acid sequence was Glu-Arg-Glu-Leu-Pro-Phe-Lys-Ala-Lys-His-Ala-Tyr-Ser-Thr-Ile. The purified enzyme had a high specificity for L -Leu- p -nitroanilide.     

A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine

A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe⁺³-bathophenanthroline colorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, 1- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The N-glucuronide has been shown to undergo acidic hydrolysis in the urine to release the free N-hydroxyarylamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the 1-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2-naphthylamine in a variety of species.     

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Fluorescence histochemical detection of hydrolases in tissue sections and culture cells

Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity and/or diffusion of the fluorescent tags. Satisfactory results are obtained for alkaline phosphatase, non-specific esterases and proteases with naphthol AS and 4-methoxy-2-naphthylamine coupled to nitrosalicylaldehyde. If, however, cultured monolayer cells are investigated, all synthetic substrates with fluorescent tags are suitable, including those that have so far only been used for biochemical hydrolase measurements. The fluorescent leaving groups are naphthol AS and its derivates, 4-methoxy-2-naphthylamine, aminomethylcoumarin, aminomethyltrifluoromethylcoumarin, methylumbelliferon, fluorescein and, with some limitations, also 1- and 2-naphthol. These fluorescence methods are more sensitive than the corresponding dye procedures. In addition, the fluorescence techniques allow the use of more synthetic substrates and therefore more information become available than with dye histochemistry about the enzymic properties of culture cells.     

Hydrolase histochemistry of the thymus: development and species variations

In the present investigation the localization and activity of alkaline, neutral, and acid hydrolases of the thymus were studied during development of rats and mice and of various adult species using histochemical methods. If different procedures of tissue pretreatment were employed, several inhibition effects and morphological as well as enzyme histochemical artifacts occurred dependent on the mode of tissue pretreatment. After embedding in glycol methacrylate, sections of the thymus showed a better structural preservation than cryostat sections but were accompanied by a drastic decrease of activity and low localization quality of the final reaction products especially in the case of protease studies with 4-methoxy-2-naphthylamine peptides as substrates. Smears of thymic cells facilitated the allocation of enzymes to mobile or fixed cells in the stroma of the thymus. The perivascular localization of aminopeptidase M could only be shown with combined techniques. In comparison, primarily the proteases yielded information on the thymic stroma and in this context especially on the epithelial reticular cells and the stroma proper but also on thymocytes (lymphocytes) and enabled a species-dependent subdivision of the thymic reticulum already in the light microscope. Enzyme histochemically the development of the rat and mouse thymus could be subdivided into an early period and perinatal (pre- and postnatal) period of functional differentiation. Morphological (proliferation of cortical lymphocytes) and enzyme histochemical changes (disappearance of dipeptidylpeptidase IV, significant loss of alkaline phosphatase activity and beginning activity increase of aminopeptidase M) occurred primarily at the transition from the early to the prenatal period. During the postnatal phase, a significant activation of lysosomal enzymes in the thymic medulla and general enzymatic differentiation of the cortical epithelial reticular cells were found. Species differences and species similarities for the respective enzymes and their localization as well as for the thymic cells were noticed for adult rats, mice, guinea-pigs, hamsters, and marmoset monkeys. Differences were true especially for the thymocytes; less species differences were seen for the epithelial reticular cells; capsular and perivascular connective tissue and the macrophages behaved rather similarly. Species-independently certain medullary epithelial reticular cells showed high and typically localized alkaline phosphatase activities and species-dependently also high activities of neutral hydrolases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in membrane-bound aminopeptidase on bone marrow-derived macrophages during their maturation in vitro

Myeloid colonies obtained by culturing mouse bone marrow cells with mouse lung conditioned medium were kept for up to 21 days in culture and the aminopeptidase content in single cells measured after staining with leucine 4-methoxy-2-naphthylamide. The enzyme was detectable only in mononuclear and not in granulocytic cells. The number of cells carrying the enzyme and the concentration of the enzyme in the mononuclear cells taken from whole dishes or single colonies increased remarkably but not uniformly from 7 days to maximal values at 13 days of culture, and then decreased again. The timing varied for individual colonies. Maximal enzyme concentrations were found in cells intermediate between the center and the fringe of a colony. However, most cells in a given colony showed concentration increases up to 13 days of culturing. During its life span in culture the mononuclear cell appears to gain aminopeptidase at the cell membrane and lose it again prior to death.     

Essential Features of the Class V Myosin from Budding Yeast for ASH1 mRNA Transport

Myo4p, a single-headed and nonprocessive class V myosin in budding yeast, transports >20 different mRNAs asymmetrically to the bud. Here, we determine the features of the Myo4p motor that are necessary for correct localization of ASH1 mRNA to the daughter cell, a process that also requires the adapter protein She3p and the dimeric mRNA-binding protein She2p. The rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA, confirming that the rod contains the primary binding site for She3p. The requirement for both the rod region and She3p can be bypassed by directly coupling the mRNA-binding protein She2p to Myo4p. ASH1 mRNA was also correctly localized when one motor was bound per dimeric She2p, or when two motors were joined together by a leucine zipper. Because multiple mRNAs are cotransported to the bud, it is likely that this process involves multiple motor transport regardless of the number of motors per zip code. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p–She3p–She2p–ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.