[41]
HUMAN LIVER AMINOPEPTIDASE
[41] H u m a n
By
495
Liver Aminopeptidase
GWYNIX'EH. LITTLE, WILLIS L. STARNES, and FRANCIS J. BEHAL
H u m a n 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 m a x i m u m when the residue is L-alanine, but certain other amino acids with nonpolar R groups, e.g., 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 m a n y investigators that H L A aetivity could be attributed to leueine aminopeptidase (LAP),1 but it is now clear t h a t H L A is chemically distinet from LAP. H L A catalyzes the general reaction: RCII(N+H3)CONHR' + It20 ~ RCH(N+H~)COO- + H3N+R ' R'N*H3 m a y be a m m o n i u m ion, an amino aeid, an oligopeptide, flnaphthylamine, or p-nitroaniline. The relative rates of hydrolysis of several substrates are shown in Table I. H L A must be assayed with a variety of substrates and under a variety of conditions, depending on TABLE I SUBSTRATE SPECIFICITYOF HUMAN LIVER AMINOPEPTIDASE Aminoaeyl-/3-naphthylamides L-Alanyl-~-naphthylamide L-Phenylalanyl-~-naphthylamide L-Methionyl-/~-naphthylamide L-Leucyl-f~-naphthylamide L-Arginyl-/~-naphthylamide L-Tryptophanyl-~-naphthylamide Glycyl-~-naphthylamide L-Lysyl-~-naphthylamide L-Seryl-/~-naphthylamide L-Threonyl-~-naphthylamide L-Glutamyl-/~-naphthylamide L-Valyl-/3-naphthylamide L-Isoleucyl-~-naphthylamide
1See this series Vol. 19 [33].
Relative rate (%) 100 62.8 57.5 36.4 28.0 19.2 13.7 13.4 6.0 5.7 3.7 2.8 2.8
Dipeptides
Relative rate (%)
L-Ala-L-Trp L-Ala-L-Val L-Ala-L-Ala L-Ala-L-Phe L-Leu-L-Leu L-Ala-L-Leu L-Leu-L-Ala L-Ala-L-Ser L-Phe-Gly L-Ala-L-Glu L-Ala-Gly L-Ala-L-Asp b-Leu-Gly L-Ile-Gly L-Ser-Gly
100 47.3 45.4 42.8 35.9 27.1 25.6 22.5 19.2 19.0 17.8 11.4 5.5 1.5 1.3
496
EXOPEPTIDASES
[41]
the state of purity of the enzyme and the specific results desired. The various applicable assay methods necessary for detailed work with this enzyme are given below. These are based, first, on the substrate used, and then on the various instrumental methods available.
Assay Methods Assay with Aminoacyl-fl-naphthylamide Substrates Colorimetric Method 2 The release of fl-naphthylamine from aminoacyl-fl-naphthylamides is determined by diazotization after a discrete incubation period. This method is useful for following the progress of purification and for laboratories without a recording spectrophotometer or fluorometer. Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 0.9 ml of buffer plus 0.5 ml of buffered substrate fl-Naphthylamine standard: 0.06 mM fl-naphthylamine Trichloroacetic acid (TCA), 2.5 M Sodium nitrite, 15 mM Ammonium sulfamate, 45 mM N-(1-Naphthyl)ethylenediamine dihydrochloride, 2 mM in 95% ethanol Procedure. Begin the reaction by adding 0.1 ml of the enzyme solution to the assay mixture at zero time and incubate with a blank (which is identical except that it lacks the enzyme solution) for 10-30 min at 37 °. At the end of the incubation period add 0.5 ml of 2.5 M TCA to the sample to stop the reaction. To the blank add 0.5 ml of 2.5 M TCA solution followed immediately by 0.1 ml of enzyme. To develop the color add 1.0 ml of sodium nitrite; after 3 min add 1.0 ml of amonium sulfamate; after 2 min add 2.0 ml of N-(1 naphthyl)ethylenediamine. Allow 'the color to develop for 30 min, then read the absorbance of the blank and sample against water at 580 nm. The quantity of fl-naphthylamine released is determined from the absorbance of the sample relative to that of a standard solution prepared by substituting 0.06 mM fl-naphthylamine for substrate in the assay mixture. 2j. A. Goldbarg and A. M. Rutenburg, Cancer 11,283 (1958).
[41]
HUMAN LIVER AMINOPEPTIDASE
497
Fluorometric M e t h o d 3
Free fl-naphthylamine is intensely fluorescent whereas aminoacyl-flnaphthylamides are only slightly fluorescent. This fact permits continuous monitoring of the progress of hydrolysis by means of a recording fluorometer or spectrofluorometer. The fluorometric assay is used for kinetic studies where maximum sensitivity is desired. Reagents
Phosphate buffer, 0.1 M , pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate (volumes may have to be adjusted for various-sized cuvettes) fl-Naphthylamine standard, 0.01 mM: assay mixture plus 0.1 ml of 0.3 mM fl-naphthylamine Procedure. Place the cuvette containing the assay mixture in the fluorometer and allow the temperature to equilibrate to 37 °. Start the reaction, and record the rate of change in fluorescence at 410 nm relative to the fl-naphthylamine standard. The excitation wavelength is 340 nm. Spectrophotometric M e t h o d 4
This assay is useful for kinetic work in the presence of compounds that interfere with the fluorometric assay. It lacks the sensitivity of the fluorometric assay, however. Reagents
Phosphate buffer, 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 nil of substrate Procedure. Pipette the reaction mixture into a 3-ml quartz cuvette and allow the temperature to equilibrate to 379. Begin the reaction by adding 0.1 ml of enzyme solution, and record the rate of increase in absorbance at 340 nm. The slope of the resulting curve is proportional to the amount of fl-naphthylamine released per unit of time. The amount may be calculated from a standard curve or from the measured extinction coefficient of the difference spectrum between L-alanyl-fl-naphthylamide and fl-naphthylamine at 340 nm under these conditions.
L. J. Greenberg, Biochem. Biophys. Res. Commun. 9, 430 (1962). 4H. Lee, J. N. LaRue, and I. B. Wilson, Anal. Biochem. 41, 397 (1971).
498
EXOPEPTIDASES
[41]
Assay with Aminoacyl-p-nitroanilide Substrates Spectrophotometric Method ~ This assay is more sensitive than the spectrophotometric assay for fl-naphthylamides owing to the higher extinction coefficient of p-nitroaniline as compared to fl-naphthylamine.
Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-p-nitroanilide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate
Procedure. Same as for fl-naphthylamides except that the rate of increase in absorbance is recorded at the absorption maximum of p-nitroaniline, 405 nm. Assay with Dipeptide and Amino Acid Amide Substrates Spectrophotometric Method Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: nonaromatic amides or dipeptides, 3.0 mM, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate
Procedure. Begin the reaction by adding 0.1 ml of enzyme to the assay mixture in a 3-ml quartz cuvette and record the decrease in absorbance at 220 nm maintaining the temperture at 37 °. The slope of the line is proportional to the amount of hydrolysis of the amide or peptide bond, and the rate in micromoles per minute may be determined from a standard curve prepared by reading the absorbance at 220 nm of mixtures of substrate and products simulating varying degrees of hydrolysis. Colorimetric Ninhydrin Method This method is based on the reaction with ninhydrin of a-amino groups or ammonia produced by hydrolysis of dipeptides or amino acid amides. The reaction can be carried out manually as described below or by means of an automatic amino acid analyzer, in which case the 5It. Tuppy, W. Wiesbauer, and E. Wintersberger, Hoppe-Seyler's Z. Physiol. Chem. 329, 278 (1962).
[41]
HUMAN LIVER AMINOPEPTIDASE
499
assay mixture is set up in test tubes as in the spectrophotometric method above. After 30 min incubation at 37 °, the reaction is stopped by placing the tubes in a boiling water bath for 10 min. The sample is then prepared for amino acid analysis by standard techniques. The automated procedure has the advantage of greater sensitivity since the products are separated from the substrate, which is also ninhydrin positive. The manual procedure is carried out as follows2
Reagents Phosphate buffer. 0.01 M, pH 6.8 Substrate: 3.0 mM dipeptide, pH 6.8 Assay mixture: 0.9 ml of buffer plus 0.5 ml of substrate Picric acid, 0.04 M Citrate-methyl Cellosolve diluent: equal volumes of 0.2 M citrate buffer, pH 5.0, and methyl Cellosolve Ninhydrin reagent: prepared by mixing 1 volume of 0.2 M citrate buffer, pH 5.0, containing 1.6 mg of stannous chloride (SnC12. 2H~O) per milliliter, with 1 volume of methyl Cellosolve, containing 40 mg of ninhydrin per milliliter Ethanol, 60%
Procedure. Prepare assay mixture in quadruplicate for each sample to be assayed. Preincubate the tubes at 37 ° for 5 rain, then begin the reaction in two of the tubes by adding 0.1 ml of enzyme solution. The other two tubes are blanks. Incubate all four tubes 10-30 min then stop the reactions by adding 2.0 ml of the picric acid solution. To the blanks add 2.0 ml of picric acid solution followed immediately by 0.1 ml of enzyme solution. To develop the color take duplicate 0.25-ml aliquots from each of the four tubes and add 0.75 ml of the citrate-methyl Cellosolve diluent and 1.25 ml of the ninhydrin solution. Heat in a boiling water bath for exactly 5 min and immediately cool in ice water. Add 8.5 ml of 60% ethanol, mix thoroughly, and read the absorbance against water at 570 rim. Micromoles of substrate hydrolyzed can be determined from a standard curve prepared by running the ninhydrin reaction on a series of mixtures of the dipeptide and its component amino acids representing varying degrees of hydrolysis. Definition of Aminopeptidase Unit and Specific Activity. One unit of aminopeptidase activity is defined as the amount of enzyme that will liberate 1 ~mole of fl-naphthylamine or of p-nitroaniline or hydrolyze G. A. Fleisher, M. Pankow, and C. Warmka, Clin. Chim. Acta 9, 254 (1964).
500
[411
EXOPEPTIDASES
TABLE II PURIFICATION OF HUMAN LIVER AMINOPEPTIDASE
Isolation step Homogenization and autolysis Ammonium sulfate fractionation Gel filtration Anion-exchange chromatography Hydroxyapatite chromatography
Total activity a (units)
Recovery (%)
Total protein (rag)
1990
100
103,000
2590
130
4,720
2560 1090
129 55
578 63.7
824
41
18.0
Specific activity a 0. 0193 0. 521 4.43 17.1 45.7
Purification factor 1 27 229 887 2370
a Units of activity and specific activity are defined in the text. 1 ~mole of an amino acid amide or dipeptide per minute under the conditions described. Specific activity is defined as units per milligram of protein.
Purification of H u m a n Liver A m i n o p e p t i d a s e H L A purified b y the following procedure behaves as a single species on acrylamide gels 7,8 and in sedimentation equilibrium experiments in denaturing solvents. 8 The results obtained at each step are summarized in T a b l e I I . The colorimetric assay with L - a l a n y l - # - n a p h t h y l a m i d e is normally used during the purification. Homogenization and Autolysis. H u m a n liver is obtained at autopsy from unembalmed cadavers and frozen at - - 2 0 ° until used. Dissect about 1000 g of liver free of fat and connective tissue, cut into small pieces, cover with phosphate buffer (0.02 M, p H 6.86), and homogenize in a Waring Blendor or similar homogenizer until thoroughly disrupted. Add enough buffer to m a k e the final volume 4 liters, then allow the homogehate to autolyze for 24 hr at 37 ° (autolysis for periods much longer than 24 hr results in loss of activity). After autolysis, the solid residue is removed by centrifugation. Washing of the pellet provides higher yields of enzyme, but the procedure is laborious. Ammonium Sulfate Fractionation. Add solid a m m o n i u m sulfate to a final concentration of 1.0 M and then pour onto a 10-cm bed of porous 7F. J. Behal, G. H. Little, and R. A. Klein, Biochem. Biophys. Acta 178, 118 (1969). W. L. Starnes and F. J. Behal, Biochemistry 13, 3221 (1974).
[41]
HUMAN LIVER AMINOPEPTIDASE
501
glass (Bio-Glas 200, 100-200 mesh, Bio-Rad Laboratories) in a large filter funnel (2-liter capacity, coarse sintered glass) equilibrated with 1.0 M ammonium sulfate. Elute with 1.0 M ammonium sulfate until protein ceases to appear in the effluent, and then begin elution with water and collect fractions containing aminopeptidase activity and concentrate by ultrafiltration to a volume of 50 ml or less. The efficiency of later steps can be increased if the Bio-Glas step is repeated, permitting omission of the hydroxyapatite step. The Bio-Glas is regenerated by washing with 0.1 M NaOH to remove bound protein followed by several washes with water and reequilibration with 1.0 M (NH~)2S04. Gel Filtration,. Apply the concentrated material from the above step to a Pharmacia K 50/100 column packed with Sephadex G-200 equilibrated with 0.1 M sodium borate buffer at pH 7.5 containing 0.5 mole of NaC] per liter. Elute the column with approximately 2000 ml of the same buffer. Pool the fractions containing aminopeptidase activity and concentrate by ultrafiltration to a volume of approximately 25 ml. Anion-Exchange Chromatography. Dialyze the ultrafiltrate against 0.005 M Tris buffer, pH 8.6, and apply it to a 2.5 X 69 cm column of DEAE-cellulosc equilibrated with the same buffer. Elute with 2000 ml of linearly increasing NaC1 gradient at pH 7.0 with an initial NaC1 concentration of 0.01 M and a limiting concentration of 0.2 M. Pool the active fractions and concentrate by ultrafiltration to a volume of about 5 ml. Hydroxyapatite Chromatography. Dialyze the ultrafiltrate against 0.001 M phosphate buffer at pH 7.0 and apply it to a 1.0 X 50 cm column of hydroxyapatite (Hypatite C: Clarkson Chemical Co.) in the same buffer. Elute the column by means of a linear 500-ml gradient of phosphate buffer at pH 7.0 with the concentration increasing from 0.01 to 0.05 M. Collect 5-ml fractions and combine and concentrate those containing aminopeptidase activity.
Properties of Human Liver Aminopeptidase HLA was earlier confused with LAP and Was so designated in the literature. HLA differs markedly from LAP in several aspects: HLA is a zinc metalloenzyme which also forms a cobalt metal-enzyme complex; HLA is far more active on leucyl-fl-naphthylamide than on leucylglycine, whereas LAP is maximally active on leucylglycine; HLA is maximally active on substrates with L-alanyl-N-terminal residues, whereas LAP is more active on substrates with L-leucyl residues; the HLA pH optimum is 6.8, rather than 8.8-9.0. HLA is one of several electrophoretically distinct "isoenzymes"
502
[41]
EXOPEPTIDASES
TABLE I I I PHYSICAL PROPEWI'IES OF HUMAN LIVER AM1NOPEPTIDASE
Property
Method
Extinction coefficient, ( E ~ %nm) Sedimentation coefficient
From dry weight and ultraviolet spcctrum Sedimentation velocity
1.75 ml-mg-'
Synthetic boundary diffusion
4.02 X 10-7 cm 2 sec -1
From amino acid content Itigh speed sedimentation equilibrium
0.730 cm a g-1 223,000 ± 17,000
Sedimentation velocity Gel filtration Sedimentation equilibrium in guanidinium chloride Sodium dodecyl sulfate gel electrophoresis
235,000 ± 10,000 242,000 ± 21,000 118,000 ± 5,000
(~0,~)
Diffusion coefficient (D2°0,w) Partial specific volumc Molecular weight of native enzyme
Subunit molecular weight
Value
10.1
)< 1 0 - l a s e e - l
120,000 ± 12,000
which occur in human blood. These have moderately broad specificity and exhibit maximum activity when the N-terminal amino acid residue is nonpolar. In contrast, other groups of aminopeptidases present in blood, liver, and other tissues are characterized by maximum activity when the N-terminal residue is acidic or basic. The other circulating "isoenzymes" of the HLA family are derived from pancreas, kidney, and duodenum2 HLA is a sialoglycoprotein containing 17.46% carbohydrate (4.32% glucosamine, 4.14% sialic acid, 9.00% hexoses). The molecular weight of the enzyme in dilute salt solutions is approximately 235,000. s,l° In denaturing solvents, HLA dissociates to a single species with molecular weight near 118,000 as determined by sedimentation equilibrium and SDS acrylamide gel electrophoresis in the presence and in the absence of 6 M urea. Earlier evidence obtained by agarose gel chromatography in guanidinium chloride indicates that the enzyme can further dissociate into smaller subunits having a molecular weight of roughly 38,000.1° This result has not been confirmed by means of sedimentation equilibrium experiments, but it is not yet excluded since there are unique problems inherent in the analysis of the physical properties of glycoproteins. 9 F. J. Behal, B. Asserson, F. Dawson, and J. Hardman, Arch Biochem. Biophys. 111, 335 (1965). 10G. H. Little and F. J. Behal, Biochim. Biophys Acta 243, 312 (1971).
[41]
HUMAN LIVER A M I N O P E P T I D A S E
503
TABLE IV CHEMICAL COMPOSITION OF HUMAN LIVER AMINOPEPTIDASE
Constituent
Residues per 118,000 dMtons
Amino acids Lysine Histidinc Arginine Tryptophan ~ Aspartic acid~ Threonine Serine Glutamie acid Proline Glycine Alanine Cysteine~ Valine~
41 19 35 31 105 54 57 96 44 43 58 tr 53
Constituent Amino acids (continued) Methionine Isoleucine'~ Leueine Tyrosine Phenylalanine Carbohydrates Glucosamine Sialic acid(s) Hexoses Metals Zinc
Residues per 118,000 daltons
16 35 81 42 36 29 16 62 1
Corrected to zero hydrolysis time. b Free acid plus amide. As cysteic acid. d Corrected to infinite hydrolysis time. H L A c o n t a i n s 8.3 -4- 1.5 n a n o a t o m s of t i g h t l y b o u n d zinc per millig r a m of p r o t e i n (i.e., one zinc ion per 118,000 d a l t o n s u b u n i t ) , is activ a t e d up to 2.4-fold b y the a d d i t i o n of Co 2÷, which binds loosely in a n o n c o m p e t i t i v e m a n n e r '1 a n d is i n h i b i t e d b y p u r o m y c i n , 1-~ penicillin,13 c e r t a i n a m i n o acids, TM peptides, 14 carboxylic acids, 1'~ a n d a l i p h a t i c amines, is I n general, a m i n o acids are n o n c o m p e t i t i v e i n h i b i t o r s of a l a n y l f l - n a p h t h y l a m i d e h y d r o l y s i s with K~ values n e a r 1.0 m M . D i p e p t i d e s g e n e r a l l y are c o m p e t i t i v e i n h i b i t o r s with s i g n i f i c a n t l y lower K~ values. Some physical properties of H L A a n d the m e t h o d b y which t h e y were o b t a i n e d are s u m m a r i z e d in T a b l e I I I . T h e chemical composition of H L A is presented in T a b l e IV.
11C. W. Garner and F. J. Behal, Biochemistry 13, 3227 (1974). ~ G. H. Little, Ph.D. Thesis, Medical College of Georgia, 1970. 1~Unpublished observation. n C. W. Garner and F. J. Behal, Biochemistry 14, 3208 (1975). ~5C. W. Garner, personal communication.
HUMAN LIVER AMINOPEPTIDASE
[41] H u m a n
By
495
Liver Aminopeptidase
GWYNIX'EH. LITTLE, WILLIS L. STARNES, and FRANCIS J. BEHAL
H u m a n 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 m a x i m u m when the residue is L-alanine, but certain other amino acids with nonpolar R groups, e.g., 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 m a n y investigators that H L A aetivity could be attributed to leueine aminopeptidase (LAP),1 but it is now clear t h a t H L A is chemically distinet from LAP. H L A catalyzes the general reaction: RCII(N+H3)CONHR' + It20 ~ RCH(N+H~)COO- + H3N+R ' R'N*H3 m a y be a m m o n i u m ion, an amino aeid, an oligopeptide, flnaphthylamine, or p-nitroaniline. The relative rates of hydrolysis of several substrates are shown in Table I. H L A must be assayed with a variety of substrates and under a variety of conditions, depending on TABLE I SUBSTRATE SPECIFICITYOF HUMAN LIVER AMINOPEPTIDASE Aminoaeyl-/3-naphthylamides L-Alanyl-~-naphthylamide L-Phenylalanyl-~-naphthylamide L-Methionyl-/~-naphthylamide L-Leucyl-f~-naphthylamide L-Arginyl-/~-naphthylamide L-Tryptophanyl-~-naphthylamide Glycyl-~-naphthylamide L-Lysyl-~-naphthylamide L-Seryl-/~-naphthylamide L-Threonyl-~-naphthylamide L-Glutamyl-/~-naphthylamide L-Valyl-/3-naphthylamide L-Isoleucyl-~-naphthylamide
1See this series Vol. 19 [33].
Relative rate (%) 100 62.8 57.5 36.4 28.0 19.2 13.7 13.4 6.0 5.7 3.7 2.8 2.8
Dipeptides
Relative rate (%)
L-Ala-L-Trp L-Ala-L-Val L-Ala-L-Ala L-Ala-L-Phe L-Leu-L-Leu L-Ala-L-Leu L-Leu-L-Ala L-Ala-L-Ser L-Phe-Gly L-Ala-L-Glu L-Ala-Gly L-Ala-L-Asp b-Leu-Gly L-Ile-Gly L-Ser-Gly
100 47.3 45.4 42.8 35.9 27.1 25.6 22.5 19.2 19.0 17.8 11.4 5.5 1.5 1.3
496
EXOPEPTIDASES
[41]
the state of purity of the enzyme and the specific results desired. The various applicable assay methods necessary for detailed work with this enzyme are given below. These are based, first, on the substrate used, and then on the various instrumental methods available.
Assay Methods Assay with Aminoacyl-fl-naphthylamide Substrates Colorimetric Method 2 The release of fl-naphthylamine from aminoacyl-fl-naphthylamides is determined by diazotization after a discrete incubation period. This method is useful for following the progress of purification and for laboratories without a recording spectrophotometer or fluorometer. Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 0.9 ml of buffer plus 0.5 ml of buffered substrate fl-Naphthylamine standard: 0.06 mM fl-naphthylamine Trichloroacetic acid (TCA), 2.5 M Sodium nitrite, 15 mM Ammonium sulfamate, 45 mM N-(1-Naphthyl)ethylenediamine dihydrochloride, 2 mM in 95% ethanol Procedure. Begin the reaction by adding 0.1 ml of the enzyme solution to the assay mixture at zero time and incubate with a blank (which is identical except that it lacks the enzyme solution) for 10-30 min at 37 °. At the end of the incubation period add 0.5 ml of 2.5 M TCA to the sample to stop the reaction. To the blank add 0.5 ml of 2.5 M TCA solution followed immediately by 0.1 ml of enzyme. To develop the color add 1.0 ml of sodium nitrite; after 3 min add 1.0 ml of amonium sulfamate; after 2 min add 2.0 ml of N-(1 naphthyl)ethylenediamine. Allow 'the color to develop for 30 min, then read the absorbance of the blank and sample against water at 580 nm. The quantity of fl-naphthylamine released is determined from the absorbance of the sample relative to that of a standard solution prepared by substituting 0.06 mM fl-naphthylamine for substrate in the assay mixture. 2j. A. Goldbarg and A. M. Rutenburg, Cancer 11,283 (1958).
[41]
HUMAN LIVER AMINOPEPTIDASE
497
Fluorometric M e t h o d 3
Free fl-naphthylamine is intensely fluorescent whereas aminoacyl-flnaphthylamides are only slightly fluorescent. This fact permits continuous monitoring of the progress of hydrolysis by means of a recording fluorometer or spectrofluorometer. The fluorometric assay is used for kinetic studies where maximum sensitivity is desired. Reagents
Phosphate buffer, 0.1 M , pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate (volumes may have to be adjusted for various-sized cuvettes) fl-Naphthylamine standard, 0.01 mM: assay mixture plus 0.1 ml of 0.3 mM fl-naphthylamine Procedure. Place the cuvette containing the assay mixture in the fluorometer and allow the temperature to equilibrate to 37 °. Start the reaction, and record the rate of change in fluorescence at 410 nm relative to the fl-naphthylamine standard. The excitation wavelength is 340 nm. Spectrophotometric M e t h o d 4
This assay is useful for kinetic work in the presence of compounds that interfere with the fluorometric assay. It lacks the sensitivity of the fluorometric assay, however. Reagents
Phosphate buffer, 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-fl-naphthylamide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 nil of substrate Procedure. Pipette the reaction mixture into a 3-ml quartz cuvette and allow the temperature to equilibrate to 379. Begin the reaction by adding 0.1 ml of enzyme solution, and record the rate of increase in absorbance at 340 nm. The slope of the resulting curve is proportional to the amount of fl-naphthylamine released per unit of time. The amount may be calculated from a standard curve or from the measured extinction coefficient of the difference spectrum between L-alanyl-fl-naphthylamide and fl-naphthylamine at 340 nm under these conditions.
L. J. Greenberg, Biochem. Biophys. Res. Commun. 9, 430 (1962). 4H. Lee, J. N. LaRue, and I. B. Wilson, Anal. Biochem. 41, 397 (1971).
498
EXOPEPTIDASES
[41]
Assay with Aminoacyl-p-nitroanilide Substrates Spectrophotometric Method ~ This assay is more sensitive than the spectrophotometric assay for fl-naphthylamides owing to the higher extinction coefficient of p-nitroaniline as compared to fl-naphthylamine.
Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: 3.0 mM L-alanyl-p-nitroanilide, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate
Procedure. Same as for fl-naphthylamides except that the rate of increase in absorbance is recorded at the absorption maximum of p-nitroaniline, 405 nm. Assay with Dipeptide and Amino Acid Amide Substrates Spectrophotometric Method Reagents Phosphate buffer: 0.1 M, pH 6.8 Substrate: nonaromatic amides or dipeptides, 3.0 mM, pH 6.8 Assay mixture: 1.9 ml of buffer plus 1.0 ml of substrate
Procedure. Begin the reaction by adding 0.1 ml of enzyme to the assay mixture in a 3-ml quartz cuvette and record the decrease in absorbance at 220 nm maintaining the temperture at 37 °. The slope of the line is proportional to the amount of hydrolysis of the amide or peptide bond, and the rate in micromoles per minute may be determined from a standard curve prepared by reading the absorbance at 220 nm of mixtures of substrate and products simulating varying degrees of hydrolysis. Colorimetric Ninhydrin Method This method is based on the reaction with ninhydrin of a-amino groups or ammonia produced by hydrolysis of dipeptides or amino acid amides. The reaction can be carried out manually as described below or by means of an automatic amino acid analyzer, in which case the 5It. Tuppy, W. Wiesbauer, and E. Wintersberger, Hoppe-Seyler's Z. Physiol. Chem. 329, 278 (1962).
[41]
HUMAN LIVER AMINOPEPTIDASE
499
assay mixture is set up in test tubes as in the spectrophotometric method above. After 30 min incubation at 37 °, the reaction is stopped by placing the tubes in a boiling water bath for 10 min. The sample is then prepared for amino acid analysis by standard techniques. The automated procedure has the advantage of greater sensitivity since the products are separated from the substrate, which is also ninhydrin positive. The manual procedure is carried out as follows2
Reagents Phosphate buffer. 0.01 M, pH 6.8 Substrate: 3.0 mM dipeptide, pH 6.8 Assay mixture: 0.9 ml of buffer plus 0.5 ml of substrate Picric acid, 0.04 M Citrate-methyl Cellosolve diluent: equal volumes of 0.2 M citrate buffer, pH 5.0, and methyl Cellosolve Ninhydrin reagent: prepared by mixing 1 volume of 0.2 M citrate buffer, pH 5.0, containing 1.6 mg of stannous chloride (SnC12. 2H~O) per milliliter, with 1 volume of methyl Cellosolve, containing 40 mg of ninhydrin per milliliter Ethanol, 60%
Procedure. Prepare assay mixture in quadruplicate for each sample to be assayed. Preincubate the tubes at 37 ° for 5 rain, then begin the reaction in two of the tubes by adding 0.1 ml of enzyme solution. The other two tubes are blanks. Incubate all four tubes 10-30 min then stop the reactions by adding 2.0 ml of the picric acid solution. To the blanks add 2.0 ml of picric acid solution followed immediately by 0.1 ml of enzyme solution. To develop the color take duplicate 0.25-ml aliquots from each of the four tubes and add 0.75 ml of the citrate-methyl Cellosolve diluent and 1.25 ml of the ninhydrin solution. Heat in a boiling water bath for exactly 5 min and immediately cool in ice water. Add 8.5 ml of 60% ethanol, mix thoroughly, and read the absorbance against water at 570 rim. Micromoles of substrate hydrolyzed can be determined from a standard curve prepared by running the ninhydrin reaction on a series of mixtures of the dipeptide and its component amino acids representing varying degrees of hydrolysis. Definition of Aminopeptidase Unit and Specific Activity. One unit of aminopeptidase activity is defined as the amount of enzyme that will liberate 1 ~mole of fl-naphthylamine or of p-nitroaniline or hydrolyze G. A. Fleisher, M. Pankow, and C. Warmka, Clin. Chim. Acta 9, 254 (1964).
500
[411
EXOPEPTIDASES
TABLE II PURIFICATION OF HUMAN LIVER AMINOPEPTIDASE
Isolation step Homogenization and autolysis Ammonium sulfate fractionation Gel filtration Anion-exchange chromatography Hydroxyapatite chromatography
Total activity a (units)
Recovery (%)
Total protein (rag)
1990
100
103,000
2590
130
4,720
2560 1090
129 55
578 63.7
824
41
18.0
Specific activity a 0. 0193 0. 521 4.43 17.1 45.7
Purification factor 1 27 229 887 2370
a Units of activity and specific activity are defined in the text. 1 ~mole of an amino acid amide or dipeptide per minute under the conditions described. Specific activity is defined as units per milligram of protein.
Purification of H u m a n Liver A m i n o p e p t i d a s e H L A purified b y the following procedure behaves as a single species on acrylamide gels 7,8 and in sedimentation equilibrium experiments in denaturing solvents. 8 The results obtained at each step are summarized in T a b l e I I . The colorimetric assay with L - a l a n y l - # - n a p h t h y l a m i d e is normally used during the purification. Homogenization and Autolysis. H u m a n liver is obtained at autopsy from unembalmed cadavers and frozen at - - 2 0 ° until used. Dissect about 1000 g of liver free of fat and connective tissue, cut into small pieces, cover with phosphate buffer (0.02 M, p H 6.86), and homogenize in a Waring Blendor or similar homogenizer until thoroughly disrupted. Add enough buffer to m a k e the final volume 4 liters, then allow the homogehate to autolyze for 24 hr at 37 ° (autolysis for periods much longer than 24 hr results in loss of activity). After autolysis, the solid residue is removed by centrifugation. Washing of the pellet provides higher yields of enzyme, but the procedure is laborious. Ammonium Sulfate Fractionation. Add solid a m m o n i u m sulfate to a final concentration of 1.0 M and then pour onto a 10-cm bed of porous 7F. J. Behal, G. H. Little, and R. A. Klein, Biochem. Biophys. Acta 178, 118 (1969). W. L. Starnes and F. J. Behal, Biochemistry 13, 3221 (1974).
[41]
HUMAN LIVER AMINOPEPTIDASE
501
glass (Bio-Glas 200, 100-200 mesh, Bio-Rad Laboratories) in a large filter funnel (2-liter capacity, coarse sintered glass) equilibrated with 1.0 M ammonium sulfate. Elute with 1.0 M ammonium sulfate until protein ceases to appear in the effluent, and then begin elution with water and collect fractions containing aminopeptidase activity and concentrate by ultrafiltration to a volume of 50 ml or less. The efficiency of later steps can be increased if the Bio-Glas step is repeated, permitting omission of the hydroxyapatite step. The Bio-Glas is regenerated by washing with 0.1 M NaOH to remove bound protein followed by several washes with water and reequilibration with 1.0 M (NH~)2S04. Gel Filtration,. Apply the concentrated material from the above step to a Pharmacia K 50/100 column packed with Sephadex G-200 equilibrated with 0.1 M sodium borate buffer at pH 7.5 containing 0.5 mole of NaC] per liter. Elute the column with approximately 2000 ml of the same buffer. Pool the fractions containing aminopeptidase activity and concentrate by ultrafiltration to a volume of approximately 25 ml. Anion-Exchange Chromatography. Dialyze the ultrafiltrate against 0.005 M Tris buffer, pH 8.6, and apply it to a 2.5 X 69 cm column of DEAE-cellulosc equilibrated with the same buffer. Elute with 2000 ml of linearly increasing NaC1 gradient at pH 7.0 with an initial NaC1 concentration of 0.01 M and a limiting concentration of 0.2 M. Pool the active fractions and concentrate by ultrafiltration to a volume of about 5 ml. Hydroxyapatite Chromatography. Dialyze the ultrafiltrate against 0.001 M phosphate buffer at pH 7.0 and apply it to a 1.0 X 50 cm column of hydroxyapatite (Hypatite C: Clarkson Chemical Co.) in the same buffer. Elute the column by means of a linear 500-ml gradient of phosphate buffer at pH 7.0 with the concentration increasing from 0.01 to 0.05 M. Collect 5-ml fractions and combine and concentrate those containing aminopeptidase activity.
Properties of Human Liver Aminopeptidase HLA was earlier confused with LAP and Was so designated in the literature. HLA differs markedly from LAP in several aspects: HLA is a zinc metalloenzyme which also forms a cobalt metal-enzyme complex; HLA is far more active on leucyl-fl-naphthylamide than on leucylglycine, whereas LAP is maximally active on leucylglycine; HLA is maximally active on substrates with L-alanyl-N-terminal residues, whereas LAP is more active on substrates with L-leucyl residues; the HLA pH optimum is 6.8, rather than 8.8-9.0. HLA is one of several electrophoretically distinct "isoenzymes"
502
[41]
EXOPEPTIDASES
TABLE I I I PHYSICAL PROPEWI'IES OF HUMAN LIVER AM1NOPEPTIDASE
Property
Method
Extinction coefficient, ( E ~ %nm) Sedimentation coefficient
From dry weight and ultraviolet spcctrum Sedimentation velocity
1.75 ml-mg-'
Synthetic boundary diffusion
4.02 X 10-7 cm 2 sec -1
From amino acid content Itigh speed sedimentation equilibrium
0.730 cm a g-1 223,000 ± 17,000
Sedimentation velocity Gel filtration Sedimentation equilibrium in guanidinium chloride Sodium dodecyl sulfate gel electrophoresis
235,000 ± 10,000 242,000 ± 21,000 118,000 ± 5,000
(~0,~)
Diffusion coefficient (D2°0,w) Partial specific volumc Molecular weight of native enzyme
Subunit molecular weight
Value
10.1
)< 1 0 - l a s e e - l
120,000 ± 12,000
which occur in human blood. These have moderately broad specificity and exhibit maximum activity when the N-terminal amino acid residue is nonpolar. In contrast, other groups of aminopeptidases present in blood, liver, and other tissues are characterized by maximum activity when the N-terminal residue is acidic or basic. The other circulating "isoenzymes" of the HLA family are derived from pancreas, kidney, and duodenum2 HLA is a sialoglycoprotein containing 17.46% carbohydrate (4.32% glucosamine, 4.14% sialic acid, 9.00% hexoses). The molecular weight of the enzyme in dilute salt solutions is approximately 235,000. s,l° In denaturing solvents, HLA dissociates to a single species with molecular weight near 118,000 as determined by sedimentation equilibrium and SDS acrylamide gel electrophoresis in the presence and in the absence of 6 M urea. Earlier evidence obtained by agarose gel chromatography in guanidinium chloride indicates that the enzyme can further dissociate into smaller subunits having a molecular weight of roughly 38,000.1° This result has not been confirmed by means of sedimentation equilibrium experiments, but it is not yet excluded since there are unique problems inherent in the analysis of the physical properties of glycoproteins. 9 F. J. Behal, B. Asserson, F. Dawson, and J. Hardman, Arch Biochem. Biophys. 111, 335 (1965). 10G. H. Little and F. J. Behal, Biochim. Biophys Acta 243, 312 (1971).
[41]
HUMAN LIVER A M I N O P E P T I D A S E
503
TABLE IV CHEMICAL COMPOSITION OF HUMAN LIVER AMINOPEPTIDASE
Constituent
Residues per 118,000 dMtons
Amino acids Lysine Histidinc Arginine Tryptophan ~ Aspartic acid~ Threonine Serine Glutamie acid Proline Glycine Alanine Cysteine~ Valine~
41 19 35 31 105 54 57 96 44 43 58 tr 53
Constituent Amino acids (continued) Methionine Isoleucine'~ Leueine Tyrosine Phenylalanine Carbohydrates Glucosamine Sialic acid(s) Hexoses Metals Zinc
Residues per 118,000 daltons
16 35 81 42 36 29 16 62 1
Corrected to zero hydrolysis time. b Free acid plus amide. As cysteic acid. d Corrected to infinite hydrolysis time. H L A c o n t a i n s 8.3 -4- 1.5 n a n o a t o m s of t i g h t l y b o u n d zinc per millig r a m of p r o t e i n (i.e., one zinc ion per 118,000 d a l t o n s u b u n i t ) , is activ a t e d up to 2.4-fold b y the a d d i t i o n of Co 2÷, which binds loosely in a n o n c o m p e t i t i v e m a n n e r '1 a n d is i n h i b i t e d b y p u r o m y c i n , 1-~ penicillin,13 c e r t a i n a m i n o acids, TM peptides, 14 carboxylic acids, 1'~ a n d a l i p h a t i c amines, is I n general, a m i n o acids are n o n c o m p e t i t i v e i n h i b i t o r s of a l a n y l f l - n a p h t h y l a m i d e h y d r o l y s i s with K~ values n e a r 1.0 m M . D i p e p t i d e s g e n e r a l l y are c o m p e t i t i v e i n h i b i t o r s with s i g n i f i c a n t l y lower K~ values. Some physical properties of H L A a n d the m e t h o d b y which t h e y were o b t a i n e d are s u m m a r i z e d in T a b l e I I I . T h e chemical composition of H L A is presented in T a b l e IV.
11C. W. Garner and F. J. Behal, Biochemistry 13, 3227 (1974). ~ G. H. Little, Ph.D. Thesis, Medical College of Georgia, 1970. 1~Unpublished observation. n C. W. Garner and F. J. Behal, Biochemistry 14, 3208 (1975). ~5C. W. Garner, personal communication.
