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The LNA-hydrolysing enzyme in the toxin of Bombina variegata
Toxrcon, 1967, Vol. 5. pp . 105-109 . Pergarno n Press Ltd., Printed in Great Britain
THE LNA-HYDROLYSING ENZYME IN THE TOXIN OF BOMBINA YARIEGATA* H. MOLZER and H. MICHL
Chemical Institute, University of Agriculture, Vienna, Austria (Acceptedfor publication 1 May 1967) Abstract-The L-leucine-ß-naphthylarnide hydrolysing enzyme occurring in the toxin of the unk Bombina variegata is characterized by substrate specificity, inhibitors and other qualities. The results show that this enzyme is different from aminopeptidases from other sources. INTRODUCTION
ENZYMES which hydrolyse L-leucine-/4-naphthylamide (LNA) were demonstrated in several amphibian and reptile toxins [1] . Their maxima of activity are between pH 7-0 and 7-5. These enzymes are not identical with the well known leucine aminopeptidases (LAP) of swine kidney, intestinal mucosa or other sources [2], which possess pH optima of approximately 9-0. The present experiments were designed to characterize the LNA-hydrolysing enzyme of the unk Bombina variegata . The data presented in this communication deal with methods for partial purification, with the substrate specificity and with inhibitors of this enzyme . MATERIALS AND METHODS
Enzyme Preparations were obtained from Bombina toxin [31 by the following single step procedures Ultrafiltration. This was performed with a `Metall Druckfiltergerät' MD 70-15 using an ultrafilter LSG 60 with a pore diameter smaller than 5 mtA (Membranfiltergesellschaft, Göttingen) . Precipitation with ammonium sulfate. A 0-5 per cent solution of Bombina toxin in Soerensen buffer pH 7-0 was precipitated stepwise with crystallized ammonium sulfate at 0°. The fraction obtained between 60-70 per cent of saturation was eight-ten times more active than the starting material. The recovery of activity was 85 per cent. Chromatography on Sephadex. Thirty mg toxin dissolved in 6 ml 0-9 per cent NaCI solution were applied to a Sephadex G-200 column (100 x 2-5 cm) equilibrated in the same solvent . The void volume of the column, determined with blue dextran, was 90 ml. A LKB fraction collector with a Uvicord Absorptiometer (2537 A) was used. Elution was performed with 0-9 per cent aqueous NaC1 solution and 3 ml samples were taken . *This communication is dedicated to Prof. Dr . F. WEssELY, Head of the Organic Department, Chemical Institute, University of Vienna, on the occasion of his seventieth birthday. 105
106
H. MOLZER and H. MICHL
The amidase as well as the phosphatases (L . Paschinger, Chemical Institute, University of Agriculture, Vienna, personal communication) and the amylase [4] were eluated after 150 ml . Arylamidase activity was increased sevenfold. DEAE cellulose chromatography. A solution of 60 mg toxin in 4 ml 0.02 M tris-buffer of pH 7-0 was placed on a column 18 x 1-5 cm and washed with 25 ml of the same buffer . Gradient elution was performed with increasing NaCl concentrations (0-02-1-1 M). Five ml samples were taken. Arylamidase was eluated by 0-08 M NaCl, ahead of the other enzymes (amylase 0-2 M, phosphatase 1-2 M). Recovery was 90 per cent ; increase in activity was 14-fold. Substrates and inhibitors Amino acid arylamides and amino acid amides . These were a gift from Dr. H.
NESVADBA
(Peptidlabor, Fa. Sanabo, Vienna) or synthetized according to the methods of BRENNER and HuBER [5] and SMITH and SLONIM [6] . Oligopeptides . These were gifts as above or commercial products (Fluka, Buchs, Switzerland) . Inhibitors . These were commercial products (Fluka, Buchs, Switzerland) ; Diisopropylfluorophosphate (Schuchart, Munich, Germany) was redistilled twice. It had no effect on LAP from swine kidney.
Electrophoresis High voltage orpolyacrylamide electrophoresis. These were performed as described
previously [7, 8] .
Assay methods Amino acid naphthylamidase activity. This assay method was done as described in a
previous paper [1].
Amino acid amidase activity. An 0-01 M solution of amino acid amide (0-5 ml) in Soerensen buffer pH 7-0 and 0-5 ml of a partially purified (Ultrafiltration) enzyme solution in the same buffer were incubated for 2 hr at 37°. After halting the reaction by boiling, samples of 2, 5,10 and 20 j,l were placed on a strip of Whatman filter paper 3MM moistened with pyridine acetate buffer pH 4-7. Electrophoresis was applied for 30 min at a potential gradient of 40 V/cm . The amino acids formed by the enzymatic action remained near the origin . The unhydrolysed amino acid amides migrated to the cathode. After the electrophoresic separation the dry filter paper strip was immersed in a cadmium acetate-ninhydrin reagent [9] After drying, the strip was heated at 80° for 15 min and TABLE 1 . RATIO OF MIORATION DISTANCES OF AMINO ACID AMIDESILYSINE
Tyrosine amide Phenylalanine amide Leucine amide Isoleucine amide Hydroxyproline amide
0.85 0.95 1-03 1 .05 1-24
Serine amide Proline amide Alanine amide Glycine amide Lysine amide
(Pyridine acetic acid-water (1 : 1 :98), pH 4-7, about 40 V/cm).
130 132 139 1-60 1-65
The LNA-hydrolysing Enzyme in the Toxin of Bombina variegata
107
then kept in a desiccator over concentrated sulfuric acid for 20 hr . The colored spots were cut out and the dye was eluated with 8 ml of methanol in stoppered glass tubes . The optical densities of the solutions were measured at 500 mp. for the amino acids and at 352 m/A for proline and hydroxyproline with a Beckman DB spectrophotometer. Quantitation was done by comparison with calibration curves . Cleavage ofpeptides. The ability of the enzyme under investigation to hydrolyse peptides was proved using paper chromatography (butanol-acetic acid-water, 4:1 :5) . Quantitative assay was done by the method mentioned above [9] . RESULTS AND DISCUSSION TABLE 2. HYDROLYSIS OF L-AMINO ACID-ß-NAPHTHYLAMIDES Leucine-p-naphthylamide Alanine naphthylamide Oxyproline naphthylamide Methionine naphthylamide Valine naphthylamide Proline naphthylamide Glycine naphthylamide Lysine naphthylamide Arginine naphthylamide Isoleucine naphthylamide
100 70 65 55 50 45 30 25 20 10
Histidine naphthylamide a-Glutamic acid naphthylamide Serine naphthylamide Phenylalanine naphthylamide Tyrosine naphthylamide
All less than 10
Cysteine naphthylamide a-Aspartic acid naphthylamide ß-Aspartic acid naphthylamide Threonine naphthylamide Tryptophane naphthylamide
Practically not hydrolysed
(The value for L-leucine-p-naphthylamide is given as 100 and for the other naphthylamides as the relative rate). TABLE 3. HYDROLYSIS OF L-AMINO ACID AMIDES AND PEPTIDES Leucine amide Alanine amide Oxyproline amide Proline amide Glycine amide Isoleucine amide Lysine amide Tyrosine amide Phenylalanine amide Serine amide Leu-gly Leu-ala Leu-tyr L-leucino-ß-naphthylamide
100 75 60 55 45 20 15 10 5 5 65 40 25 25
(The value for L-leucine amide is given as 100 and for the following substrates as the relative rate).
Il . MOLZEIZ and H . MICIIL
108
The following peptides were hydrolysed : Leu-gly-gly, gly-met, gly-leu-leu, gly-leu-gly, gly-val. Cleavage could not be ascertained for the peptides : Ala-phe, gly-ser, gly-alâ, ala-his, gly-aspNH Z. Incubation of the partially purified enzyme for several hours at 37" with a hexapeptide Ala-glu-his-phe-ala-aspNH Z occurring in the toxin of Bombina variegata [3] yields alanine in traces only. Action of the enzyme on a directly haemolysing peptide of the same toxin [10] seems to destroy its haemolytic activity after a prolonged time (storing of toxin solution at room temperature, ultrafiltration of concentrated toxin solution) [11] . ß-Naphthylamides and amides were practically not hydrolysed when functional groups or aromatic or heterocyclic rings were attached to the ß-carbon of the amino acid. The substrate specificity as shown in Tables 2 and 3 differs greatly from substrate specificities of aminopeptidases prepared from various kinds of tissues of rat as well as from homogenates of dog, and human livers . It is also different from the specificity of purified aminopeptidase from swine kidney [12] . The molar ratio of hydrolysed L-leucine amide/hydrolysed LNA was approximately 4 : 1 . This is considerably less than in the case of LAP from swine kidney where the ratio is 10,000-20,000 : 1 [13]. By ammonium sulfate precipitation it can be shown that a trypsin like activity is precipitated at 50 per cent saturation whereas the hydrolysing activity on the other substrates precipitates at 70 per cent saturation, thus indicating the presence of two distinct enzymes. The trypsin like activity (pH optimum 7), responsible for hydrolysing lysine-and arginine naphthylamide, cannot be inhibited by trypsin inhibitor from pancreas . It is increased only twofold by the precipitation procedure . It may be mentioned that the optimal pH for hydrolysing L-proline-tg-naphthylamide is 8-5 against 7-0-7-5 for the other hydrolysed substrates. It was, however, not possible to separate a proline naphthylamidase activity from the LNAse by chromatographic or gel electrophoretic methods. Also, amidase and arylamidase activities could not be separated using the same methods. It should be noted that the activity of the arylamide hydrolysing enzyme of the unfractionated toxin of Bombina variegata is rather weak, which may be illustrated by the following figures : at 37° it takes 0-3 mg of lyophilised toxin 4 hr to completely hydrolyse 0-29 mg of L-leucine-ß-naphthylamide . At 20° this activity will drop to 45 per cent of its 37° value, just reaching 10 per cent at 10°. The enzyme is stable to temperature and solution in distilled water for an extended period of time. TABLC 4
Hgz+ Cul+ Pbz+ Znz+ Cdz+ Ni2 + Col+ M nz+ Mg2+
1-3 2-5 5.0 1-6 2.5 7-9 6-3 5-0 7-9
x x x x x x x x x
10 - ' 10-° 10-1 10 - ' 10-1 10-1 10- z 10- z 10-1
p-Chloromercuribenzoate Diisopropylfluorophosphate Citrate 8-Hydroxychinoline ß-Mercaptoethanol EDTA
4 . 0 x 10-'
2-5 7-9 7-9 1-6 3-2
x x x >: x
10 -1 10-1 10-3 10-3 10 - z
The LNA-hydrolysing Enzyme in the Toxin of Bonchina variegata
109
To find the inhibitor concentration in M/l at 50 percent inhibition of LNAse activity (Table 4), incubation mixtures were used containing 0-1 mg of lyophilised toxin per 2 ml 0-02 M tris buffer pH 7-0 plus 0-5 ml of 0002 M LNA solution and 1 ml of inhibitor solution (10- s to 10-' M). Smaller concentrations of Mn$+ (approximately 10-; M), Mg$ ` (2-5 .'< 10-; M) and ß-mercaptoethanol (2-5 x 10-5 M) have a slight activating action . However, dialysis against distilled water for 14 hr have no effect on the enzyme's activity. The enzyme of Bombina toxin is practically not inhibited by versene, but strongly so by P-chloromercuribenzoate, thus differing strikingly from swine kidney aminopeptidase and from an aminopeptidase found to effect the moulting of nematode parasites [14] . The influence of metal ions resembles somewhat that on LAP from swine kidney .
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] (I1] [12] [13] [14]
REFERENCES
MICHL, H. and Mot.zelt, H., The occurrence of L-leucyl-p-naphthylamide (LNA) splitting enzymes in some amphibia and reptile venoms . Toxicon 2, 281, 1965 . SMITH, E. L. and HILL, R. L. : The Enzymes. New York : Academic Press, Vol. 4, 1960 . Kiss, G. und MICHL, H., Über das Giftsekret der Gelbbauchunke, Bombina variegata L. Toxicon 1, 33, 1962 . MICHL, H. und PAsruszYN, A., Vergleichende Untersuchungen von Amylasen . Mh . Chem . 3, 978, 1964. BRENNER, M. und HUBER, W., Herstellung von a-Aminosdureestem durch Alkoholyse der Methylester. Helv. chim. Acta 36,1109, 1953 . SMrrH, E. L. and SLoNUI, N. B., Leucineaminopeptidase. J. biol. Chem . 176, 835, 1948 . MICHL, H., High voltage electrophoresis . Chromat. Rev. 1, 11, 1959. MICHL, H. und PAsruszyN, A., Über Zonenschürfung bei der Gelelektrophorese. Mikrochim . Acta 5, 880, 1963 . HEILMANN, J., BAROLLMR, J. und WATZKE, E., Beitragzur Aminosäurebestimmung auf Papierchromatogrammen. Hoppe-Seyler's Z. physiol. Chem . 309, 219, 1957 . BACHMAYER, H., Ph .D. Thesis, University of Vienna, Chemical Institute, 1966 . LIDAUER, W., Dipl . Ing. Thesis, Chemical Institute, University of Agriculture, Vienna, 1967 . NACHLAS, M. M., GOLIsiEm, T. P. and SE.iGMAN, A. M., An evaluation of aminopeptidase specifity with seven chromogenic substrates . Archs biochem. Biophys . 97, 223, 1962 . PAITERSON, K. E., HSIAO, S. H. and KEPPEL, A., Studies on dipeptidases and aminopeptidases. J. biol. Chem . 238, 3611 . 1963 . RoGERs, W. P., The role of leucineaminopeptidase in the moulting of nematode parasite ;. Comp . Biochem . Physiol. 14, 311, 1965 .
THE LNA-HYDROLYSING ENZYME IN THE TOXIN OF BOMBINA YARIEGATA* H. MOLZER and H. MICHL
Chemical Institute, University of Agriculture, Vienna, Austria (Acceptedfor publication 1 May 1967) Abstract-The L-leucine-ß-naphthylarnide hydrolysing enzyme occurring in the toxin of the unk Bombina variegata is characterized by substrate specificity, inhibitors and other qualities. The results show that this enzyme is different from aminopeptidases from other sources. INTRODUCTION
ENZYMES which hydrolyse L-leucine-/4-naphthylamide (LNA) were demonstrated in several amphibian and reptile toxins [1] . Their maxima of activity are between pH 7-0 and 7-5. These enzymes are not identical with the well known leucine aminopeptidases (LAP) of swine kidney, intestinal mucosa or other sources [2], which possess pH optima of approximately 9-0. The present experiments were designed to characterize the LNA-hydrolysing enzyme of the unk Bombina variegata . The data presented in this communication deal with methods for partial purification, with the substrate specificity and with inhibitors of this enzyme . MATERIALS AND METHODS
Enzyme Preparations were obtained from Bombina toxin [31 by the following single step procedures Ultrafiltration. This was performed with a `Metall Druckfiltergerät' MD 70-15 using an ultrafilter LSG 60 with a pore diameter smaller than 5 mtA (Membranfiltergesellschaft, Göttingen) . Precipitation with ammonium sulfate. A 0-5 per cent solution of Bombina toxin in Soerensen buffer pH 7-0 was precipitated stepwise with crystallized ammonium sulfate at 0°. The fraction obtained between 60-70 per cent of saturation was eight-ten times more active than the starting material. The recovery of activity was 85 per cent. Chromatography on Sephadex. Thirty mg toxin dissolved in 6 ml 0-9 per cent NaCI solution were applied to a Sephadex G-200 column (100 x 2-5 cm) equilibrated in the same solvent . The void volume of the column, determined with blue dextran, was 90 ml. A LKB fraction collector with a Uvicord Absorptiometer (2537 A) was used. Elution was performed with 0-9 per cent aqueous NaC1 solution and 3 ml samples were taken . *This communication is dedicated to Prof. Dr . F. WEssELY, Head of the Organic Department, Chemical Institute, University of Vienna, on the occasion of his seventieth birthday. 105
106
H. MOLZER and H. MICHL
The amidase as well as the phosphatases (L . Paschinger, Chemical Institute, University of Agriculture, Vienna, personal communication) and the amylase [4] were eluated after 150 ml . Arylamidase activity was increased sevenfold. DEAE cellulose chromatography. A solution of 60 mg toxin in 4 ml 0.02 M tris-buffer of pH 7-0 was placed on a column 18 x 1-5 cm and washed with 25 ml of the same buffer . Gradient elution was performed with increasing NaCl concentrations (0-02-1-1 M). Five ml samples were taken. Arylamidase was eluated by 0-08 M NaCl, ahead of the other enzymes (amylase 0-2 M, phosphatase 1-2 M). Recovery was 90 per cent ; increase in activity was 14-fold. Substrates and inhibitors Amino acid arylamides and amino acid amides . These were a gift from Dr. H.
NESVADBA
(Peptidlabor, Fa. Sanabo, Vienna) or synthetized according to the methods of BRENNER and HuBER [5] and SMITH and SLONIM [6] . Oligopeptides . These were gifts as above or commercial products (Fluka, Buchs, Switzerland) . Inhibitors . These were commercial products (Fluka, Buchs, Switzerland) ; Diisopropylfluorophosphate (Schuchart, Munich, Germany) was redistilled twice. It had no effect on LAP from swine kidney.
Electrophoresis High voltage orpolyacrylamide electrophoresis. These were performed as described
previously [7, 8] .
Assay methods Amino acid naphthylamidase activity. This assay method was done as described in a
previous paper [1].
Amino acid amidase activity. An 0-01 M solution of amino acid amide (0-5 ml) in Soerensen buffer pH 7-0 and 0-5 ml of a partially purified (Ultrafiltration) enzyme solution in the same buffer were incubated for 2 hr at 37°. After halting the reaction by boiling, samples of 2, 5,10 and 20 j,l were placed on a strip of Whatman filter paper 3MM moistened with pyridine acetate buffer pH 4-7. Electrophoresis was applied for 30 min at a potential gradient of 40 V/cm . The amino acids formed by the enzymatic action remained near the origin . The unhydrolysed amino acid amides migrated to the cathode. After the electrophoresic separation the dry filter paper strip was immersed in a cadmium acetate-ninhydrin reagent [9] After drying, the strip was heated at 80° for 15 min and TABLE 1 . RATIO OF MIORATION DISTANCES OF AMINO ACID AMIDESILYSINE
Tyrosine amide Phenylalanine amide Leucine amide Isoleucine amide Hydroxyproline amide
0.85 0.95 1-03 1 .05 1-24
Serine amide Proline amide Alanine amide Glycine amide Lysine amide
(Pyridine acetic acid-water (1 : 1 :98), pH 4-7, about 40 V/cm).
130 132 139 1-60 1-65
The LNA-hydrolysing Enzyme in the Toxin of Bombina variegata
107
then kept in a desiccator over concentrated sulfuric acid for 20 hr . The colored spots were cut out and the dye was eluated with 8 ml of methanol in stoppered glass tubes . The optical densities of the solutions were measured at 500 mp. for the amino acids and at 352 m/A for proline and hydroxyproline with a Beckman DB spectrophotometer. Quantitation was done by comparison with calibration curves . Cleavage ofpeptides. The ability of the enzyme under investigation to hydrolyse peptides was proved using paper chromatography (butanol-acetic acid-water, 4:1 :5) . Quantitative assay was done by the method mentioned above [9] . RESULTS AND DISCUSSION TABLE 2. HYDROLYSIS OF L-AMINO ACID-ß-NAPHTHYLAMIDES Leucine-p-naphthylamide Alanine naphthylamide Oxyproline naphthylamide Methionine naphthylamide Valine naphthylamide Proline naphthylamide Glycine naphthylamide Lysine naphthylamide Arginine naphthylamide Isoleucine naphthylamide
100 70 65 55 50 45 30 25 20 10
Histidine naphthylamide a-Glutamic acid naphthylamide Serine naphthylamide Phenylalanine naphthylamide Tyrosine naphthylamide
All less than 10
Cysteine naphthylamide a-Aspartic acid naphthylamide ß-Aspartic acid naphthylamide Threonine naphthylamide Tryptophane naphthylamide
Practically not hydrolysed
(The value for L-leucine-p-naphthylamide is given as 100 and for the other naphthylamides as the relative rate). TABLE 3. HYDROLYSIS OF L-AMINO ACID AMIDES AND PEPTIDES Leucine amide Alanine amide Oxyproline amide Proline amide Glycine amide Isoleucine amide Lysine amide Tyrosine amide Phenylalanine amide Serine amide Leu-gly Leu-ala Leu-tyr L-leucino-ß-naphthylamide
100 75 60 55 45 20 15 10 5 5 65 40 25 25
(The value for L-leucine amide is given as 100 and for the following substrates as the relative rate).
Il . MOLZEIZ and H . MICIIL
108
The following peptides were hydrolysed : Leu-gly-gly, gly-met, gly-leu-leu, gly-leu-gly, gly-val. Cleavage could not be ascertained for the peptides : Ala-phe, gly-ser, gly-alâ, ala-his, gly-aspNH Z. Incubation of the partially purified enzyme for several hours at 37" with a hexapeptide Ala-glu-his-phe-ala-aspNH Z occurring in the toxin of Bombina variegata [3] yields alanine in traces only. Action of the enzyme on a directly haemolysing peptide of the same toxin [10] seems to destroy its haemolytic activity after a prolonged time (storing of toxin solution at room temperature, ultrafiltration of concentrated toxin solution) [11] . ß-Naphthylamides and amides were practically not hydrolysed when functional groups or aromatic or heterocyclic rings were attached to the ß-carbon of the amino acid. The substrate specificity as shown in Tables 2 and 3 differs greatly from substrate specificities of aminopeptidases prepared from various kinds of tissues of rat as well as from homogenates of dog, and human livers . It is also different from the specificity of purified aminopeptidase from swine kidney [12] . The molar ratio of hydrolysed L-leucine amide/hydrolysed LNA was approximately 4 : 1 . This is considerably less than in the case of LAP from swine kidney where the ratio is 10,000-20,000 : 1 [13]. By ammonium sulfate precipitation it can be shown that a trypsin like activity is precipitated at 50 per cent saturation whereas the hydrolysing activity on the other substrates precipitates at 70 per cent saturation, thus indicating the presence of two distinct enzymes. The trypsin like activity (pH optimum 7), responsible for hydrolysing lysine-and arginine naphthylamide, cannot be inhibited by trypsin inhibitor from pancreas . It is increased only twofold by the precipitation procedure . It may be mentioned that the optimal pH for hydrolysing L-proline-tg-naphthylamide is 8-5 against 7-0-7-5 for the other hydrolysed substrates. It was, however, not possible to separate a proline naphthylamidase activity from the LNAse by chromatographic or gel electrophoretic methods. Also, amidase and arylamidase activities could not be separated using the same methods. It should be noted that the activity of the arylamide hydrolysing enzyme of the unfractionated toxin of Bombina variegata is rather weak, which may be illustrated by the following figures : at 37° it takes 0-3 mg of lyophilised toxin 4 hr to completely hydrolyse 0-29 mg of L-leucine-ß-naphthylamide . At 20° this activity will drop to 45 per cent of its 37° value, just reaching 10 per cent at 10°. The enzyme is stable to temperature and solution in distilled water for an extended period of time. TABLC 4
Hgz+ Cul+ Pbz+ Znz+ Cdz+ Ni2 + Col+ M nz+ Mg2+
1-3 2-5 5.0 1-6 2.5 7-9 6-3 5-0 7-9
x x x x x x x x x
10 - ' 10-° 10-1 10 - ' 10-1 10-1 10- z 10- z 10-1
p-Chloromercuribenzoate Diisopropylfluorophosphate Citrate 8-Hydroxychinoline ß-Mercaptoethanol EDTA
4 . 0 x 10-'
2-5 7-9 7-9 1-6 3-2
x x x >: x
10 -1 10-1 10-3 10-3 10 - z
The LNA-hydrolysing Enzyme in the Toxin of Bonchina variegata
109
To find the inhibitor concentration in M/l at 50 percent inhibition of LNAse activity (Table 4), incubation mixtures were used containing 0-1 mg of lyophilised toxin per 2 ml 0-02 M tris buffer pH 7-0 plus 0-5 ml of 0002 M LNA solution and 1 ml of inhibitor solution (10- s to 10-' M). Smaller concentrations of Mn$+ (approximately 10-; M), Mg$ ` (2-5 .'< 10-; M) and ß-mercaptoethanol (2-5 x 10-5 M) have a slight activating action . However, dialysis against distilled water for 14 hr have no effect on the enzyme's activity. The enzyme of Bombina toxin is practically not inhibited by versene, but strongly so by P-chloromercuribenzoate, thus differing strikingly from swine kidney aminopeptidase and from an aminopeptidase found to effect the moulting of nematode parasites [14] . The influence of metal ions resembles somewhat that on LAP from swine kidney .
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] (I1] [12] [13] [14]
REFERENCES
MICHL, H. and Mot.zelt, H., The occurrence of L-leucyl-p-naphthylamide (LNA) splitting enzymes in some amphibia and reptile venoms . Toxicon 2, 281, 1965 . SMITH, E. L. and HILL, R. L. : The Enzymes. New York : Academic Press, Vol. 4, 1960 . Kiss, G. und MICHL, H., Über das Giftsekret der Gelbbauchunke, Bombina variegata L. Toxicon 1, 33, 1962 . MICHL, H. und PAsruszYN, A., Vergleichende Untersuchungen von Amylasen . Mh . Chem . 3, 978, 1964. BRENNER, M. und HUBER, W., Herstellung von a-Aminosdureestem durch Alkoholyse der Methylester. Helv. chim. Acta 36,1109, 1953 . SMrrH, E. L. and SLoNUI, N. B., Leucineaminopeptidase. J. biol. Chem . 176, 835, 1948 . MICHL, H., High voltage electrophoresis . Chromat. Rev. 1, 11, 1959. MICHL, H. und PAsruszyN, A., Über Zonenschürfung bei der Gelelektrophorese. Mikrochim . Acta 5, 880, 1963 . HEILMANN, J., BAROLLMR, J. und WATZKE, E., Beitragzur Aminosäurebestimmung auf Papierchromatogrammen. Hoppe-Seyler's Z. physiol. Chem . 309, 219, 1957 . BACHMAYER, H., Ph .D. Thesis, University of Vienna, Chemical Institute, 1966 . LIDAUER, W., Dipl . Ing. Thesis, Chemical Institute, University of Agriculture, Vienna, 1967 . NACHLAS, M. M., GOLIsiEm, T. P. and SE.iGMAN, A. M., An evaluation of aminopeptidase specifity with seven chromogenic substrates . Archs biochem. Biophys . 97, 223, 1962 . PAITERSON, K. E., HSIAO, S. H. and KEPPEL, A., Studies on dipeptidases and aminopeptidases. J. biol. Chem . 238, 3611 . 1963 . RoGERs, W. P., The role of leucineaminopeptidase in the moulting of nematode parasite ;. Comp . Biochem . Physiol. 14, 311, 1965 .