Francis J. Behal

A study of human tissue aminopeptidase components

Abstract The aminopeptidase activity of human liver, small intestine, and pancreas was studied. Liver contained two chromatographically resolvable types of aminopeptidase, one Mn++-dependent and active on dipeptides and a second with no metal ion requirement and active on amino acid β-naphthylamides (BNA). Intestine contained a Mn++-dependent aminopeptidase active on dipeptides and a Co++-dependent aminopeptidase active on amino acid BNA which eluted simultaneously from a column. Pancreas also contained both types of aminopeptidase activity; the activity on amino acid BNA, Co++-dependent, was chromatographically resolved into two components. In all cases aminopeptidase activity on dipeptides was insensitive to puromycin while aminopeptidase activity on amino acid BNA was quite sensitive to puromycin.

[41] Human liver aminopeptidase

Publisher Summary Human liver aminopeptidase (HLA) catalyzes the hydrolysis of N-terminal amino acid residues from peptides, amino aeid amides, or certain ehromogenie synthetic substrates. The rate of hydrolysis is maximum when the residue is L-alanine, but certain other amino acids with nonpolar R groups, for example, L-leueine, are hydrolyzed at significant rates. Since one of the first commonly available synthetie substrates for aminopeptidases was L-leueyl-fl-naphthylamide, it was initially assumed by many investigators that HLA aetivity could be attributed to leueine aminopeptidase (LAP), but it is now clear that HLA is chemically distinct from LAP. HLA must be assayed with a variety of substrates and under a variety of conditions, depending on the state of purity of the enzyme and the specific results desired. This chapter details the various applicable assay methods necessary for detailed work with this enzyme. These are based, first, on the substrate used, and then on the various instrumental methods available. The following assay methods have been discussed in this chapter: Assay with Aminoacyl-fl-naphthylamide Substrates (Colorimetric Method, Fluorometric Method, Spectrophotometric Method), Assay with Aminoacyl-p-nitroanilide Substrates ( Spectrophotometric Method) Assay with Dipeptide and Amino Acid Amide Substrates (Spectrophotometric method, Colorimetric Ninhydrin Method). It also discusses Purification of Human Liver Aminopeptidase.

Separation and characterization of aminopeptidase and arylamidase components of human liver

Abstract Cell-free extracts of human liver were found to contain an enzyme with activity and properties very similar to swine kidney LAP; however, the enzyme was chromatographically and electrophoretically distinguishable from swine kidney LAP. The substrate specificity pattern of this liver aminopeptidase was quite similar to that of LAP, leucine residues of dipeptides or amino acid β-naphthylamides being the ones most rapidly hydrolyzed. A second enzyme in these liver extracts was found to be extremely active upon a number of amino acid β-naphthylamides but not upon leucylglycine, and was designated arylamidase. This second enzyme was most active on alanine β-naphthylamide. These two liver enzymes also differed quite markedly in pH optima, response to inhibitors, and response to divalent cation activation.

Arylamidase of human liver

Abstract 1. 1. Arylamidase from human liver, which catalyzes the hydrolysis of certain amino acid derivatives of β-naphthylamine, was purified 2120-fold by salt fractionation, gel filtration, ion-exchange chromatography, and adsorption chromatography. 2. 2. Acrylamide gel electrophoresis and analytical ultracentrifugation of the final enzyme preparation indicated that it was a single protein with a s 20, w value of 8.5. 3. 3. Only α-amino acid β-naphthylamines of the l configuration were susceptible to arylamide catalyzed hydrolysis; alanine-β-naphthylamine had the highest max value. Several other substrates in which the amino acid residue had a larger non-polar or basic R group such as methionine-β-naphthylamine or arginine-β-naphthylamine had the lower K m values. 4. 4. This enzyme had no dipeptidyl arylamidase activity, but rather cleaved amino acid residues from dipeptide-β-naphthylamine in a step-wise manner beginning with the N-terminal residue.

Calf intestinal 5′-nucleotidase

Abstract The 5′-nucleotidase activity isolated from calf intestinal mucosa has been resolved into three fractions by DEAE-cellulose column chromatography. One of these fractions was obtained free of nonspecific phosphatase activity and was used for further studies. This enzyme has maximal activity between pH 6.0–6.5 in Tris-acetate buffer and at 8.0 in sodium diethyl barbiturate buffer. Complete inactivation of the enzyme occurs in the presence of 0.001 m EDTA. Manganese ions enhance the activity to the greatest extent although this metal ion can be essentially replaced with Co ++ or Mg ++ . Further studies showed a high specificity for the hydrolysis of 5′-mononucleotides. K m values determined at pH 8.0 were 5.0–5.8 × 10 −5 m for AMP and 5.0–8.0 × 10 −5 m for GMP. At pH 6.5 the K m value for AMP is in the range 14–20 × 10 −5 m .

Studies on aminopeptidases in human blood

Abstract The aminopeptidase activity of human plasma has been shown to be a composite of several nonspecific aminopeptidase components. The properties of these components have been determined and special studies have been made on the major plasma aminopeptidase component, alpha 1 . The Michaelis constants of this component for the three substrates, l -alanyl-β-naphthamide, l -leucyl-β-naphthamide, and glycyl-β-naphthamide, are respectively, 2 × 10 −6 , 6 × 10 −5 , and 2 × 10 −2 M . The energy of activation for the hydrolysis of the leucine substrate by the alpha component is 11.5 kcal./mole.

A study of the activity of human plasma aminopeptidase components on dipeptides and chromogenic substrates

Abstract Seven chromatographically distinguishable enzymes that have aminopeptidase (AP) activity have been demonstrated in human plasma. Only four of these AP components also hydrolyze amino acid β-naphthylamides, at a rate comparable to the dipeptide hydrolytic rate. None of these components are Mn ++ -, Zn ++ -, Co ++ -, or Mg ++ -dependent. It has been shown that the structure of the N -terminal, but not the carboxyterminal, amino acid of the dipeptide substrates influences the rate of hydrolysis. One of these AP components also has high esterase activity, but the others have no esterase activity. These components differ markedly from the major erythrocyte AP component which is greatly stimulated by Zn ++ .

Arylamidase of human kidney.

Abstract Human kidney arylamidase which catalyzes the hydrolysis of certain amino acid derivatives of β-naphthylamine (βNA) was purified 520 fold by column chromatography. Acrylamide gel electrophoresis of the purified enzyme indicated that it was a single protein. Only N-unsubstituted α-amino acid-β-naphthylamides of the l -configuration were susceptible to kidney arylamidase catalyzed hydrolysis which was divalent cation dependent and had a pH optimum of 6.8. Alanine-βNA had the highest v max value. Several other substrates in which the amino acid residue had a larger nonpolar or basic R group such as methionine-βNA or arginine-βNA had the lower K m values. This enzyme had no dipeptidyl arylamidase activity, but rather cleaved amino acid residues from dipeptide-βNA in step-wise manner beginning with the N-terminal residue. This kidney enzyme appears in the urine in substantial amounts. In the urine of males a second arylamidase isozyme not of kidney origin, but apparently derived from prostate gland or seminal fluid, also appears in large amounts.

Arylamidase of human duodenum

Abstract Arylamidase from human duodenum has been prepared free of contaminating leucine aminopeptidase. It has a pH optimum of 6.8, is maximally activated by cobalt ion and hydrolyzes l -alanine-β-naphthylamide most rapidly although l -leucine-β-naphthylamide binds more firmly to the enzyme. The l configuration is required in the N-terminal residue of the substrate. This arylamidase is exopeptidic in nature since it catalyzes a stepwise hydrolysis of dipeptide-β-naphthylamide, beginning with the N-terminal residue, to amino acid and β-naphthylamide and does not split dipeptide-β-naphthylamide to yield dipeptide and β-naphthylamine.

Heterogeneity of calf intestinal alkaline phosphatase

Abstract Alkaline phosphatase activity prepared from calf intestine has been resolved into several chromatographically distinct enzyme components with DEAE-cellulose column chromatography. The presence of multiple enzyme components with distinct electrophoretic mobilities was also demonstrated by subjecting portions of the column eluate to vertical starch-gel electrophoresis. Further differentiating characteristics between the isolated enzyme components were found to be present when studying the relative activities of these enzymes on several phosphomonoester substrates. The sensitivities of the enzyme components towards heat, however, were found to be similar.