- Email: [email protected]
Determination of Branched-Chain l -Amino-Acid Aminotransferase Activity
[3]
ANALYSISOF BCAA AMINOTRANSFERASE
23
IpOHA is a potent inhibitor of KARI. 42'43At the MIC in S. typhimurium TV105 (1 /zM), the cellular levels of the keto acid precursors of the branched-chain amino acids drop below detectable levels (Table II and data not shown14). The intracellular concentrations of both substrates of KARI, acetolactate and acetohydroxybutyrate, increase by about 10-fold within 20 min, from 0.39 to 4.3 mM and from 0.05 to 0.43 mM, respectively.TM In this case, too, the feedback controls in the metabolic network lead to an increase in the levels of 2-ketobutyrate (Table II), which may also play a role in the toxicity of IpOHA. 14 The observations described above, some of which were quite unexpected, have led to reassessment of several assumptions concerning the branched-chain pathway. The analyses of intracellular metabolites described here have proved themselves to be valuable tools for opening new insights into metabolic function. Acknowledgment This work was supported in part by Grant 338/92 from the Israel Science Foundation. 42 A. Aulabaugh and J. V. Schloss, J. Cell Biol. 107, 402A (1988). 43 j. V. Schloss and A. Aulabangh, in "Biosynthesis of Branched Chain Amino Acids" (Z. Barak, D. M. Chipman, and J. V. Schloss, eds.), pp. 329-356. VCH, Weinheim, Germany, 1990.
[3] D e t e r m i n a t i o n o f B r a n c h e d - C h a i n L - A m i n o - A c i d Aminotransferase Activity B y P E T E R SCHADEWALDT
Introduction The first step in the catabolism of branched-chain L-amino acids is reversible transamination, which is catalyzed by branched-chain L-aminoacid aminotransferase(s) (BCAA-AT; EC 2.6.1.42). Activity and (subcellular) distribution of the enzyme has been most extensively studied in the rat. Reportedly, (iso)enzyme I (mBCAA-AT) prevails in the mitochondria of most organs and tissues. (Iso)enzyme II (leucine-methionine specific) is found in rodent liver only, and the cytosolic (iso)enzyme (cBCAAAT) is expressed mainly in brain, placenta, and ovary. Information on the
METHODS IN ENZYMOLOGY,VOL 324
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24
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
enzymes from other mammals is less abundant. 1-4 The early findings of Goto et al. 5 on (iso)enzyme distribution in human tissues have been extended. Suryawan et al. 6 performed enzyme activity and m R N A analyses and revealed considerable differences in organ distribution and established the clear predominance of mBCAA-AT in all human tissues studied with the exception of brain. Aminotransferases play an important role in the partitioning of amino acid carbon skeletons into anabolic and catabolic processes. Unlike the rather specific L-alanine (EC 2.6.1.2) and L-aspartate (EC 2.6.1.1) aminotransferases, BCAA-AT exhibits a rather broad substrate specificity. Under physiological conditions, four branched-chain L-amino acids, including Lalloisoleucine, L-glutamate, as well as the derived 2-oxo acids, may be used as substrates. Thus, competition among the various compounds may be of physiological significance for the regulation of the overall metabolism of branched-chain compounds) '2'4'6 For measurement of BCAA-AT activity, radiochemical, colorimetric, and fluorimetric end-point methods have been described. 7q° All these methods are reliable, but somewhat laborious, and none allows monitoring of the course of the transamination reaction. The coupled enzymatic assay procedures described below were developed in order to allow continuous and sensitive spectrophotometric determination of BCAA-AT activity, ha2 Assay Using Branched-Chain L-lInino Acid Substrate Principle o f M e t h o d
This assay is based on the transamination of L-leucine in the presence of 2-oxoglutarate by branched-chain L-amino-acid aminotransferase (BCAA1 A.inIchihara, "Transaminases" (P. Christen and E. E. Metzler, eds.), pp. 433-439. John Wiley & Sons, New York, 1985. 2 A. E. Harper, R. H. Miller, and K. P. Block, Annu. Rev. Nutr. 3, 409 (1984). 3 S. M. Hutson and T. R. Hall, J. Biol. Chem. 268, 3084 (1993). 4 T. R. Hall, R. WaUin, G. D. Reinhard, and S. M. Hutson, J. Biol. Chem. 268, 3092 (1993). s M. Goto, H. Shinno, and A. Ichihara, Gann 68, 663 (1977). 6 A. Suryawan, J. W. Hayes, R. A. Harris, Y. Shimomura, A. E. Jenkins, and S. Hutson, Am. J. Clin. Nutr. 68, 72 (1998). 7 A. Ichihara and E. Koyama, J. Biochem. 59, 160 (1966). 8 C. S. Hintz, W. R. Turk, N. Chambon, H. B. Bruch, P. M. Nemeth, and O. H. Lowry, Anal, Biochem. 146, 418 (1970). 9 A. Akabaysahi and T. Kato, Anal. Biochem. 182, 129 (1989). lo R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 24L 4391 (1966). 11 p. Schadewaldt, W. Hummel, U. Wendel, and F. Adelmeyer, Anal. Biochem. 230,199 (1995). 12p. Schadewaldt and F. Adelmeyer, Anal. Biochem. 238, 65 (1996).
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
IBC' 'AT I
L-Leucine~
25
4-Methyl-2-oxopentanoate~2-Hydroxy-4-methylpentanoate
2-OxoglutarateL-Glutamate
NADH
NAD+
SCHEME 1
AT), yielding 4-methyl-2-oxopentanoate. The rate of formation of the 2-oxo acid is specifically monitored in a coupled enzymatic reaction. NAD ÷dependent D-2-hydroxyisocaproate dehydrogenase (HIC-DH) is used as a coupling enzyme and the rate of decrease in NADH absorbance is continuously measured spectrophotometrically at 334 nm (Scheme 1). Materials and Methods Reagents and Solutions
Tris buffer: Prepare Tris-HCl buffer, pH 8.4, containing 0.2 M Tris; adjust the pH with HC1 solution (2 M); at - 2 0 ° this buffer is stable for months Leu solution: 85 mM L-leucine 2-OG solution: 250 mM 2-oxoglutarate, sodium salt (Merck, Rahway, N J); freshly prepared NADH solution: 4 mM, prepare daily PLP solution: 2 mM pyridoxal phosphate (Boehringer Mannheim, Indianapolis, IN); store protected from light in suitable portions at - 2 0 ° (stable for months) KIC solution: 42 mM 4-methyl-2-oxopentanoate, sodium salt (Sigma, St. Louis, MO) HIC-DH working solution: D-2-Hydroxyisocaproate dehydrogenase is not commercially available. A suitable -40-fold purified preparation in 50% glycerol (w/w) can be obtained from disrupted cells of Lactobacillus casei ssp. pseudoplantarum by a two-step liquid-liquid extraction using polyethylene glycol 1540 and polyethylene glycol 10000 followed by DEAE-cellulose chromatography (DE52; Whatman, Clifton, NJ) described by Hummel et al. 13 Adjust the enzyme activity to give a final activity concentration of 10 mM min -1 (see below); store in suitable portions at - 2 0 ° (stable for months) Enzyme dissolution buffer: Potassium phosphate buffer, 50 mM, pH 7.4 13W. Hummel, H. Schtitte, and M.-R. Kula, J. Appl. Microbiol. Biotechnol. 21, 7 (1985).
26
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
Check of Coupling Enzyme Activity. The activity of HIC-DH decreases considerably at pH >8. u Therefore, the enzyme working solution should be checked as follows. Dilute the working solution 1:100 with enzyme dissolution buffer. Enzyme activity is assessed in a 10-mm cuvette at 25 ° by measuring spectrophotometrically the N A D H consumption at 334 nm against time. The assays comprise (1-ml final volume) the following: 0.5 ml of Tris buffer, 0.05 ml each of NADH, PLP, and KIC solution, 0.1 ml of Leu solution, and 0.25 ml of H20. The reaction is started by addition of 0.05 ml of diluted HIC-DH. The test should yield a linear decrease of absorbance of 0.031 &A min -1 (___-10%). Analysis of Branched-Chain L-Amino-Acid Aminotransferase Activity. Enzyme activity is assessed at 25° in a 10-mm cuvette and a reaction volume of 1 ml by measuring spectrophotometrically the decrease in N A D H absorption at 334 nm against time. The cuvette contains 0.5 ml of Tris buffer, 0.05 ml each of NADH, PLP, and HIC-DH working solution, and up to 0.25 ml of a (crude) BCAA-AT preparation. When necessary, an appropriate volume of H20 is added to give 0.90 ml. After recording the baseline, the reaction is started by addition of 0.1 ml of Leu solution. Coupling enzyme activity in the assay should be in at least 25-fold excess. Therefore, either the sample volume should be reduced or, alternatively, the BCAA-AT preparation diluted appropriately when the linear change in absorbance exceeds 0.12 AA min-L For assessment of reagent blanks, BCAA-AT in the assay is replaced by an appropriate volume of H20. After correction of the measured absorbance change per minute for baseline and reagent blank, BCAA-AT activity concentration of the preparation is calculated by the formula [BCAA-AT] (in/~M min -1) = (AA m i n - ~ ( 1 ~ \ 6.22 / \ V s ] ×103 with Vs the sample volume in the cuvette in milliliters.
Performance of Method Precision and Accuracy. In this assay, the rates of N A D H consumption (equivalent to 4-methyl-2-oxopentanoate production from L-leucine) and of L-glutamate formation (from 2-oxoglutarate) are equivalent, indicating that stoichiometry is fully met under these conditions. Furthermore, practically identical results were obtained when enzymatic measurements were compared with data from an independent radiochemical assay, u Absorbance changes of <0.001 &A rain -1 are reliably measurable. Thus, the sensitivity of the method for detection of BCAA-AT activity is <-0.1 IzM min-L The assay is linear up to a BCAA-AT activity concentration of
[3]
ANALYSISOF BCAA AMINOTRANSFERASE
27
about 20/zM min -1. When determined with rat heart BCAA-AT activities of 1, 7.5, and 14.5/zM min -1 in the assay, the coefficient of variation within the run amounts to 7.9, 2.1, and 2.2%, respectively (n = 7). 11 The present test conditions have been optimized for analysis of rat heart mitochondrial BCAA-AT and allow measurement of the reaction rate at 0.9Vma~ for this enzyme. It may be nescessary to introduce some modifications in order to optimize the assay for (iso)enzymes from other species. Temperature Conversion Factors. When related to the rates obtained with partially purified BCAA-AT preparations from rat heart at 25 °, estimated temperature conversion factors for 20, 30, and 37° amounted to 0.7, 1.4, and 2.0, respectively (n = 3). 11 Comments. Because of the kinetic properties of HIC-DH from L. casei ssp. pseudoplantarum, 13 the present assay is largely limited to L-leucine as a substrate. However, studies including the other branched-chain L-amino acids may be performed with HIC-DH activities from other sources that exhibit the desired substrate properties, e,g., the enzyme from Lactobacillus curvatus. 14 Based on our experience, no valid measurements in tissue and cell homogenates can be performed in the presence of ammonia. Presumably, these interferences are due to the presence of L-glutamate dehydrogenase (EC 1.4.1.3), which also exhibits some L-leucine dehydrogenase activityJ 5 Therefore, it is essential to remove NH4 ÷ from the samples when necessary, e.g., by dialysis.
Assays Using Branched-Chain 2-Oxo Acid
Substrates
Principle of Methods These methods are based on the branched-chain L-amino-acid aminotransferase (BCAA-AT)-dependent transamination of 4-methyl-2-oxopentanoate in the presence of L-glutamate. The formation of 2-oxoglutarate is coupled either to L-aspartate aminotransferase (ASAT) plus L-malate dehydrogenase (MDH) (BAM assay procedure) or, alternatively, to Lalanine aminotransferase (ALAT) plus L-lactate dehydrogenase (LDH) (BAL assay procedure) as indicator systems. The transamination rate is continuously monitored by spectrophotometric measurement of the decrease in NADH absorbance at 334 nm over time (Scheme 2).
14W. Hummel, H. Schtitte, and M.-R. Kula, J. Appl. Microbiol. Biotechnol. 28, 433 (1988). 15E. L. Smith, B. M. Austen, K. M. Blumenthal, and J. F. Nyc, in "The Enzymes" (P. D. Boyer, ed.), Vol. 11, 3rd Ed., pp. 293-367. Academic Press, New York, 1975.
28
SUBSTRATE PREPARATION, ENZYME ASSAYS, 1NHIBITORS
NADH
L-Alanine
/-'i
L-Glutamate
[3]
NAD+
,ruv.
L-Lactate
2-Oxoglutarate
4-Methyl-
2-oxopentanoate
L-Glutamate
L-Aspartate ~ ' ~
L-Leucine
2-Oxoglutarate
~ Oxaloaeetate NADH
L-Malate
NAD +
SCHEME2
Materials and Methods Reagents and Solutions
The following are required for the BAM assay procedure. BAM buffer: Prepare Tris-Glu-Asp-NaOH buffer, pH 8.3, containing 0.2 M Tris, 0.6 M L-glutamic acid, 0.4 M e-aspartic acid; adjust pH with NaOH solution (2 M); stable for months at -20 ° KIC solution: 42 mM 4-methyl-2-oxopentanoate, sodium salt (Sigma); store in suitable portions at -20°; stable for months NADH solution: 4 raM, prepare daily PLP solution: 2 mM pyridoxal phosphate (Boehringer Mannheim); store protected from light in suitable portions at -20°; stable for months Enzyme dissolution buffer: potassium phosphate buffer, 100 mM, pH 7.4 ASAT working solution: Dissolve lyophilized L-aspartate aminotransferase (EC 2.6.1.1, from porcine heart; Sigma) in enzyme dissolution
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
29
buffer to give a final activity concentration of 10 mM min-l; store in suitable portions at -20°; stable for weeks MDH working solution: Dilute L-malate dehydrogenase preparation in 50% glycerol (EC 1.1.1.37, from pig heart; Boehringer Mannheim) with enzyme dissolution buffer to give a final activity concentration of 20 mM rain-l; prepare daily 2-OG solution: 250 mM 2-oxoglutarate, sodium salt (Merck), freshly prepared O A A solution: 20 mM oxaloacetate, sodium salt (Merck), freshly prepared The following are required for the BAL assay procedure. BAL buffer: Prepare Tris-Glu-Ala-NaOH buffer, pH 8.3, containing 0.2 M Tris, 0.6 M L-glutamic acid, and 0.4 M L-alanine, and adjust the pH with NaOH solution (2 M); stable for months at - 2 0 ° ALAT working solution: Dissolve lyophilized L-alanine aminotransferase (EC 2.6.1.2, from porcine heart; Sigma) in enzyme dissolution buffer to give a final activity concentration of 10 mM min-1; store in suitable portions at -20 ° (stable for weeks) LDH working solution: Dilute L-lactate dehydrogenase preparation in 50% glycerol (EC 1.1.1.27, from pig heart; Boehringer Mannheim) with enzyme dissolution buffer to give a final activity concentration of 20 mM min-1; prepare daily Pyruvate solution: 40 mM pyruvate, sodium salt (Merck), freshly prepared Solutions of KIC, NADH, PLP, 2-OG, and enzyme dilution buffer: Prepare solutions as described above for the BAM assay procedure Check of Coupling Enzyme Activities. The coupling enzyme working solutions may be checked as follows. Dilute the working solutions 1 : 100 with enzyme dissolution buffer. Enzyme activities are then assessed in a 10-mm cuvette at 25° by measuring spectrophotometrically the NADH consumption at 334 nm against time. The assays comprise (1-ml final volume) the following: for ASAT (specifications for ALAT in parentheses): 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of KIC, NADH, PLP, 2-OG, and MDH (LDH) working solution, and 0.2 ml of 1--I20.The reaction is started by addition of 0.05 ml of ASAT (ALAT); for MDH (specifications for LDH in parentheses): 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of KIC, NADH, PLP, and OAA (pyruvate) solution, and 0.25 ml of H20. The reaction is started by addition of 0.05 ml of diluted MDD (LDH) solution. Under these conditions, the diluted ASAT (ALAT) preparation should yield a linear decrease in absorbance of 0.031 AA min -1, and the diluted MDH (LDH) preparation should yield an absorbance change of 0.062 &A min -~ (___-10%).
30
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
Branched-Chain L-Amino-Acid Aminotransferase Measurement Procedure. Enzyme activity is assessed at 25° in a 10-ram cuvette and a reaction volume of 1 ml by measuring spectrophotometrically the linear decrease in NADH absorption at 334 nm against time. For the BAM assay procedure (specifications for the BAL assay in parentheses), the cuvette contains 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of NADH, PLP, ASAT (ALAT), and MDH (LDH) working solution, and up to 0.25 ml of a (crude) BCAA-AT preparation. When necessary, an appropriate volume of H20 is added to give 0.95 ml. After recording the baseline, the reaction is started by addition of 0.05 ml of KIC solution. When the linear decrease of absorbance exceeds 0.15 AA rain -1, it is advisable to reduce the sample volume or to dilute the BCAA-AT preparation appropriately. For the preparation of additional reagent blanks, the BCAA-AT in the assay is replaced by an appropriate volume of H20. The latter determination is essential in the case of the BAL assay procedure, because 4-methyl-2-oxopentanoate is also a (although poor) substrate for L-lactate dehydrogenase. After correction of the measured linear change of absorbance per minute for baseline and reagent blank, the BCAA-AT activity concentration of the preparation is calculated by the formula given above.
Performance of Methods Precision and Accuracy. When stoichiometry was checked in the BAM assay, the rates of NADH consumption and L-leucine formation were found to be equivalent. When comparative BCAA-AT activity measurements were performed with the BAM and BAL assays, practically identical results were obtained (linear regression analysis: y = 0.41 + 0.93x, r = 0.998, n = 10). In addition, a good linear correlation was found between the BAM assay and a procedure using e-leucine as substrate and o-2-hydroxyisocaproate dehydrogenase as a coupling enzyme (see above). 12 As already noted, the sensitivity of the spectrophotometric assay methods for detection of BCAA-AT activity is better than 0.1/zM min -1. The assays are linear up to a BCAA-AT activity of about 25 ~M min -1. When determined with rat heart BCAA-AT activities of 4.5, 9.7, and 23.0/~M rain -1 in the BAM assays, the coefficient of variation (within run) amounted to 2.3, 1.0, and 1.4%, respectively (n = 5). 12 Temperature Conversion Factors. When related to the rates obtained with partially purified BCAA-AT preparations from rat heart in the BAM assay at 25°, estimated temperature conversion factors for 30 and 37° were 1.4 and 2.3, respectively (n = 6)) z
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
31
Comments. The present assays are not restricted to 4-methyl-2-oxopentanoate as substrate. Other branched-chain 2-oxo acids as well as straightchain 2-oxo acids can be used as well. Thus, the assays are of general usefulness when studies of the substrate properties of BCAA-AT (iso)enzymes are to be conducted. 12 The present assay conditions have been optimized for analysis of rat heart mitochondrial BCAA-AT. According to the available kinetic data, the reaction rate should amount to 0.9 Vm~,for this enzyme. Some modifications may be needed when the test conditions are to be optimized for analysis of (iso)enzymes from other sources. As with the other NADH-dependent assay procedure using L-leucine as substrate (described above), NH4 + may cause interferences in the BAM and BAL assay procedures and must be omitted from the assay.
Sample Preparation Procedures for the extraction and purification of branched-chain Lamino-acid aminotransferase activity have been described (see Refs. 16 and 17). When the procedure described here is to be used in crude tissue extracts, laboratory animals should be heparinized and the organs thoroughly rinsed, e.g., by perfusion with ice-cold saline (0.154 M NaC1), prior to extraction in order to remove interfering hemoglobin. With rat hearts, the following extraction procedure is applied: After rinsing by retrograde perfusion, the blood-free organ is frozen with liquid N2 and pulverized in a percussion mortar, and the powder is extracted by treatment with a 10-fold volume (w/v) of potassium phosphate buffer (200 mM, pH 6.0, 5 mM EDTA) and centrifugation. For extraction of bovine aorta endothelial cells, microcarrier cultures at confluency are washed three times with a twofold volume of ice-cold phosphate-buffered saline, and cell-free extracts are then prepared by addition of an equal volume of extraction buffer [50 mM Tris-HC1 (pH 7.4), 2mM EDTA, 0.2% (v/v) Triton X-100] and centrifugation. 11'12The latter extraction procedure has also been applied to cultured human skin fibroblasts. Tissue Activities and Kinetic Properties Branched-chain L-amino-acid aminotransferase activities have been reported for a number of tissues from various mammalian species 1-4 and for 16Z. K. Korpela, Methods Enzymol. 166, 269 (1988). 17 R. Kido, Methods Enzymol. 166, 275 (1988).
32
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
One study compared enzyme activities in rat, human, and monkey tissue and found considerable tissue and species differences. 6 Because numerous, quite different assay conditions have been applied it is hardly possible, however, to compare the enzyme activity data obtained in different laboratories (see Refs. 12 and 18 for discussion and references). The human mitochondrial (iso)enzyme, quite comparable to enzymes from other mammalian sources, exhibits a rather broad substrate specificity. 1'a8 With L-leucine (1 mM), the activity against amino group acceptors (forward reaction) was in the order 2-oxoglutarate -> branched-chain > straight-chain 2-oxo acids (C3-C8). With 4-methyl-2-oxopentanoate (1 mM) the activity against amino group donors (reverse reaction) was in the order L-glutamate ~ branched-chain > straight chain (C3-C6) > other L-amino acids) 8 With respect to the kinetic properties of the enzyme, systematic studies using adequate methodology 19 appear not to have been published. Our studies of the Michaelis constants of the rat heart mitochondrial (iso)enzyme yielded the following results (Km,app., means ___ asymptotic standard errors): with u-leucine and 2-oxoglutarate as substrates, 0.30 ___ 0.02 and 0.68 _ 0.03 mM, respectively; with branched-chain 2-oxo acid substrates and L-glutamate (Kin,app. for the amino group donor in parentheses): 4-methyl-2-oxopentanoate (KIC), 0.14 _+ 0.01 (6.65 ___ 0.34) mM; 3methyl-2-oxobutanoate (KIV), 0.11 _ 0.01 (3.60 --- 0.28) mM; (R,S)-3methyl-2-oxopentanoate [(R,S)-KMV], 0.07 _ 0.01 (2.45 _ 0.19); Ki,app. values amounted to 2.10 --- 0.18, 4.19 _ 0.74, and 1.57 --- 0.21 mM, respectively. H,12 Using cell-free extracts from cultured fibroblasts, the following Km,app. values were found for the human mitochondrial (iso)enzyme2°: forward reaction with 2-oxoglutarate (Km,~pp. for the common amino group acceptor in parentheses): e-Leu, 0.62 _+ 0.04 (3.8 --+ 0.2) mM; u-Val, 2.96 -- 0.11 (4.0 _ 0.1) mM; e-Ile, 0.56 ___ 0.03 (5.5 ___ 0.2) mM; L-alloisoleucine, 1.54--- 0.07 (2.4 ---+ 0.1) mM. For 2-oxoglutarate, the estimated Ki,~pp was >60 mM. For the reverse reaction with eglutamate (Km,app for the common amino group donor in parentheses) Kin,app. estimates were as follows: KIC, 0.53 ___ 0.03 (28.3 _-_ 1.6) mM; KIV, 0.61 --- 0.03 (10.4 ___ 0.5) mM; (S)-KMV, 0.17 _ 0.02 (21.3 --- 2.3) mM; (R)-KMV, 0.06 _+ 0.01 (4.5 --- 0.5) mM. Estimated Ki,~pp. values with branched-chain 2-oxo acid substrates were ->-5 mM. No inhibitory effects of L-amino acids were noted. c u l t u r e d cells. 12,x8
18p. Schadewaldt, U. Wendel, and H.-W. Hammen, Amino Acids 9, 147 (1995). 19 R. D. Allison and D. L. Purich, Methods Enzymol. 63, 3 (1979). 20 p. Schadewaldt and H.-W. Hammen, unpublished data, 1999.
ANALYSISOF BCAA AMINOTRANSFERASE
23
IpOHA is a potent inhibitor of KARI. 42'43At the MIC in S. typhimurium TV105 (1 /zM), the cellular levels of the keto acid precursors of the branched-chain amino acids drop below detectable levels (Table II and data not shown14). The intracellular concentrations of both substrates of KARI, acetolactate and acetohydroxybutyrate, increase by about 10-fold within 20 min, from 0.39 to 4.3 mM and from 0.05 to 0.43 mM, respectively.TM In this case, too, the feedback controls in the metabolic network lead to an increase in the levels of 2-ketobutyrate (Table II), which may also play a role in the toxicity of IpOHA. 14 The observations described above, some of which were quite unexpected, have led to reassessment of several assumptions concerning the branched-chain pathway. The analyses of intracellular metabolites described here have proved themselves to be valuable tools for opening new insights into metabolic function. Acknowledgment This work was supported in part by Grant 338/92 from the Israel Science Foundation. 42 A. Aulabaugh and J. V. Schloss, J. Cell Biol. 107, 402A (1988). 43 j. V. Schloss and A. Aulabangh, in "Biosynthesis of Branched Chain Amino Acids" (Z. Barak, D. M. Chipman, and J. V. Schloss, eds.), pp. 329-356. VCH, Weinheim, Germany, 1990.
[3] D e t e r m i n a t i o n o f B r a n c h e d - C h a i n L - A m i n o - A c i d Aminotransferase Activity B y P E T E R SCHADEWALDT
Introduction The first step in the catabolism of branched-chain L-amino acids is reversible transamination, which is catalyzed by branched-chain L-aminoacid aminotransferase(s) (BCAA-AT; EC 2.6.1.42). Activity and (subcellular) distribution of the enzyme has been most extensively studied in the rat. Reportedly, (iso)enzyme I (mBCAA-AT) prevails in the mitochondria of most organs and tissues. (Iso)enzyme II (leucine-methionine specific) is found in rodent liver only, and the cytosolic (iso)enzyme (cBCAAAT) is expressed mainly in brain, placenta, and ovary. Information on the
METHODS IN ENZYMOLOGY,VOL 324
Copyright© 2000by Academic Press All fightsof reproductionin any form reserved. 0076-6879/00 $30.00
24
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
enzymes from other mammals is less abundant. 1-4 The early findings of Goto et al. 5 on (iso)enzyme distribution in human tissues have been extended. Suryawan et al. 6 performed enzyme activity and m R N A analyses and revealed considerable differences in organ distribution and established the clear predominance of mBCAA-AT in all human tissues studied with the exception of brain. Aminotransferases play an important role in the partitioning of amino acid carbon skeletons into anabolic and catabolic processes. Unlike the rather specific L-alanine (EC 2.6.1.2) and L-aspartate (EC 2.6.1.1) aminotransferases, BCAA-AT exhibits a rather broad substrate specificity. Under physiological conditions, four branched-chain L-amino acids, including Lalloisoleucine, L-glutamate, as well as the derived 2-oxo acids, may be used as substrates. Thus, competition among the various compounds may be of physiological significance for the regulation of the overall metabolism of branched-chain compounds) '2'4'6 For measurement of BCAA-AT activity, radiochemical, colorimetric, and fluorimetric end-point methods have been described. 7q° All these methods are reliable, but somewhat laborious, and none allows monitoring of the course of the transamination reaction. The coupled enzymatic assay procedures described below were developed in order to allow continuous and sensitive spectrophotometric determination of BCAA-AT activity, ha2 Assay Using Branched-Chain L-lInino Acid Substrate Principle o f M e t h o d
This assay is based on the transamination of L-leucine in the presence of 2-oxoglutarate by branched-chain L-amino-acid aminotransferase (BCAA1 A.inIchihara, "Transaminases" (P. Christen and E. E. Metzler, eds.), pp. 433-439. John Wiley & Sons, New York, 1985. 2 A. E. Harper, R. H. Miller, and K. P. Block, Annu. Rev. Nutr. 3, 409 (1984). 3 S. M. Hutson and T. R. Hall, J. Biol. Chem. 268, 3084 (1993). 4 T. R. Hall, R. WaUin, G. D. Reinhard, and S. M. Hutson, J. Biol. Chem. 268, 3092 (1993). s M. Goto, H. Shinno, and A. Ichihara, Gann 68, 663 (1977). 6 A. Suryawan, J. W. Hayes, R. A. Harris, Y. Shimomura, A. E. Jenkins, and S. Hutson, Am. J. Clin. Nutr. 68, 72 (1998). 7 A. Ichihara and E. Koyama, J. Biochem. 59, 160 (1966). 8 C. S. Hintz, W. R. Turk, N. Chambon, H. B. Bruch, P. M. Nemeth, and O. H. Lowry, Anal, Biochem. 146, 418 (1970). 9 A. Akabaysahi and T. Kato, Anal. Biochem. 182, 129 (1989). lo R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 24L 4391 (1966). 11 p. Schadewaldt, W. Hummel, U. Wendel, and F. Adelmeyer, Anal. Biochem. 230,199 (1995). 12p. Schadewaldt and F. Adelmeyer, Anal. Biochem. 238, 65 (1996).
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
IBC' 'AT I
L-Leucine~
25
4-Methyl-2-oxopentanoate~2-Hydroxy-4-methylpentanoate
2-OxoglutarateL-Glutamate
NADH
NAD+
SCHEME 1
AT), yielding 4-methyl-2-oxopentanoate. The rate of formation of the 2-oxo acid is specifically monitored in a coupled enzymatic reaction. NAD ÷dependent D-2-hydroxyisocaproate dehydrogenase (HIC-DH) is used as a coupling enzyme and the rate of decrease in NADH absorbance is continuously measured spectrophotometrically at 334 nm (Scheme 1). Materials and Methods Reagents and Solutions
Tris buffer: Prepare Tris-HCl buffer, pH 8.4, containing 0.2 M Tris; adjust the pH with HC1 solution (2 M); at - 2 0 ° this buffer is stable for months Leu solution: 85 mM L-leucine 2-OG solution: 250 mM 2-oxoglutarate, sodium salt (Merck, Rahway, N J); freshly prepared NADH solution: 4 mM, prepare daily PLP solution: 2 mM pyridoxal phosphate (Boehringer Mannheim, Indianapolis, IN); store protected from light in suitable portions at - 2 0 ° (stable for months) KIC solution: 42 mM 4-methyl-2-oxopentanoate, sodium salt (Sigma, St. Louis, MO) HIC-DH working solution: D-2-Hydroxyisocaproate dehydrogenase is not commercially available. A suitable -40-fold purified preparation in 50% glycerol (w/w) can be obtained from disrupted cells of Lactobacillus casei ssp. pseudoplantarum by a two-step liquid-liquid extraction using polyethylene glycol 1540 and polyethylene glycol 10000 followed by DEAE-cellulose chromatography (DE52; Whatman, Clifton, NJ) described by Hummel et al. 13 Adjust the enzyme activity to give a final activity concentration of 10 mM min -1 (see below); store in suitable portions at - 2 0 ° (stable for months) Enzyme dissolution buffer: Potassium phosphate buffer, 50 mM, pH 7.4 13W. Hummel, H. Schtitte, and M.-R. Kula, J. Appl. Microbiol. Biotechnol. 21, 7 (1985).
26
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
Check of Coupling Enzyme Activity. The activity of HIC-DH decreases considerably at pH >8. u Therefore, the enzyme working solution should be checked as follows. Dilute the working solution 1:100 with enzyme dissolution buffer. Enzyme activity is assessed in a 10-mm cuvette at 25 ° by measuring spectrophotometrically the N A D H consumption at 334 nm against time. The assays comprise (1-ml final volume) the following: 0.5 ml of Tris buffer, 0.05 ml each of NADH, PLP, and KIC solution, 0.1 ml of Leu solution, and 0.25 ml of H20. The reaction is started by addition of 0.05 ml of diluted HIC-DH. The test should yield a linear decrease of absorbance of 0.031 &A min -1 (___-10%). Analysis of Branched-Chain L-Amino-Acid Aminotransferase Activity. Enzyme activity is assessed at 25° in a 10-mm cuvette and a reaction volume of 1 ml by measuring spectrophotometrically the decrease in N A D H absorption at 334 nm against time. The cuvette contains 0.5 ml of Tris buffer, 0.05 ml each of NADH, PLP, and HIC-DH working solution, and up to 0.25 ml of a (crude) BCAA-AT preparation. When necessary, an appropriate volume of H20 is added to give 0.90 ml. After recording the baseline, the reaction is started by addition of 0.1 ml of Leu solution. Coupling enzyme activity in the assay should be in at least 25-fold excess. Therefore, either the sample volume should be reduced or, alternatively, the BCAA-AT preparation diluted appropriately when the linear change in absorbance exceeds 0.12 AA min-L For assessment of reagent blanks, BCAA-AT in the assay is replaced by an appropriate volume of H20. After correction of the measured absorbance change per minute for baseline and reagent blank, BCAA-AT activity concentration of the preparation is calculated by the formula [BCAA-AT] (in/~M min -1) = (AA m i n - ~ ( 1 ~ \ 6.22 / \ V s ] ×103 with Vs the sample volume in the cuvette in milliliters.
Performance of Method Precision and Accuracy. In this assay, the rates of N A D H consumption (equivalent to 4-methyl-2-oxopentanoate production from L-leucine) and of L-glutamate formation (from 2-oxoglutarate) are equivalent, indicating that stoichiometry is fully met under these conditions. Furthermore, practically identical results were obtained when enzymatic measurements were compared with data from an independent radiochemical assay, u Absorbance changes of <0.001 &A rain -1 are reliably measurable. Thus, the sensitivity of the method for detection of BCAA-AT activity is <-0.1 IzM min-L The assay is linear up to a BCAA-AT activity concentration of
[3]
ANALYSISOF BCAA AMINOTRANSFERASE
27
about 20/zM min -1. When determined with rat heart BCAA-AT activities of 1, 7.5, and 14.5/zM min -1 in the assay, the coefficient of variation within the run amounts to 7.9, 2.1, and 2.2%, respectively (n = 7). 11 The present test conditions have been optimized for analysis of rat heart mitochondrial BCAA-AT and allow measurement of the reaction rate at 0.9Vma~ for this enzyme. It may be nescessary to introduce some modifications in order to optimize the assay for (iso)enzymes from other species. Temperature Conversion Factors. When related to the rates obtained with partially purified BCAA-AT preparations from rat heart at 25 °, estimated temperature conversion factors for 20, 30, and 37° amounted to 0.7, 1.4, and 2.0, respectively (n = 3). 11 Comments. Because of the kinetic properties of HIC-DH from L. casei ssp. pseudoplantarum, 13 the present assay is largely limited to L-leucine as a substrate. However, studies including the other branched-chain L-amino acids may be performed with HIC-DH activities from other sources that exhibit the desired substrate properties, e,g., the enzyme from Lactobacillus curvatus. 14 Based on our experience, no valid measurements in tissue and cell homogenates can be performed in the presence of ammonia. Presumably, these interferences are due to the presence of L-glutamate dehydrogenase (EC 1.4.1.3), which also exhibits some L-leucine dehydrogenase activityJ 5 Therefore, it is essential to remove NH4 ÷ from the samples when necessary, e.g., by dialysis.
Assays Using Branched-Chain 2-Oxo Acid
Substrates
Principle of Methods These methods are based on the branched-chain L-amino-acid aminotransferase (BCAA-AT)-dependent transamination of 4-methyl-2-oxopentanoate in the presence of L-glutamate. The formation of 2-oxoglutarate is coupled either to L-aspartate aminotransferase (ASAT) plus L-malate dehydrogenase (MDH) (BAM assay procedure) or, alternatively, to Lalanine aminotransferase (ALAT) plus L-lactate dehydrogenase (LDH) (BAL assay procedure) as indicator systems. The transamination rate is continuously monitored by spectrophotometric measurement of the decrease in NADH absorbance at 334 nm over time (Scheme 2).
14W. Hummel, H. Schtitte, and M.-R. Kula, J. Appl. Microbiol. Biotechnol. 28, 433 (1988). 15E. L. Smith, B. M. Austen, K. M. Blumenthal, and J. F. Nyc, in "The Enzymes" (P. D. Boyer, ed.), Vol. 11, 3rd Ed., pp. 293-367. Academic Press, New York, 1975.
28
SUBSTRATE PREPARATION, ENZYME ASSAYS, 1NHIBITORS
NADH
L-Alanine
/-'i
L-Glutamate
[3]
NAD+
,ruv.
L-Lactate
2-Oxoglutarate
4-Methyl-
2-oxopentanoate
L-Glutamate
L-Aspartate ~ ' ~
L-Leucine
2-Oxoglutarate
~ Oxaloaeetate NADH
L-Malate
NAD +
SCHEME2
Materials and Methods Reagents and Solutions
The following are required for the BAM assay procedure. BAM buffer: Prepare Tris-Glu-Asp-NaOH buffer, pH 8.3, containing 0.2 M Tris, 0.6 M L-glutamic acid, 0.4 M e-aspartic acid; adjust pH with NaOH solution (2 M); stable for months at -20 ° KIC solution: 42 mM 4-methyl-2-oxopentanoate, sodium salt (Sigma); store in suitable portions at -20°; stable for months NADH solution: 4 raM, prepare daily PLP solution: 2 mM pyridoxal phosphate (Boehringer Mannheim); store protected from light in suitable portions at -20°; stable for months Enzyme dissolution buffer: potassium phosphate buffer, 100 mM, pH 7.4 ASAT working solution: Dissolve lyophilized L-aspartate aminotransferase (EC 2.6.1.1, from porcine heart; Sigma) in enzyme dissolution
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
29
buffer to give a final activity concentration of 10 mM min-l; store in suitable portions at -20°; stable for weeks MDH working solution: Dilute L-malate dehydrogenase preparation in 50% glycerol (EC 1.1.1.37, from pig heart; Boehringer Mannheim) with enzyme dissolution buffer to give a final activity concentration of 20 mM rain-l; prepare daily 2-OG solution: 250 mM 2-oxoglutarate, sodium salt (Merck), freshly prepared O A A solution: 20 mM oxaloacetate, sodium salt (Merck), freshly prepared The following are required for the BAL assay procedure. BAL buffer: Prepare Tris-Glu-Ala-NaOH buffer, pH 8.3, containing 0.2 M Tris, 0.6 M L-glutamic acid, and 0.4 M L-alanine, and adjust the pH with NaOH solution (2 M); stable for months at - 2 0 ° ALAT working solution: Dissolve lyophilized L-alanine aminotransferase (EC 2.6.1.2, from porcine heart; Sigma) in enzyme dissolution buffer to give a final activity concentration of 10 mM min-1; store in suitable portions at -20 ° (stable for weeks) LDH working solution: Dilute L-lactate dehydrogenase preparation in 50% glycerol (EC 1.1.1.27, from pig heart; Boehringer Mannheim) with enzyme dissolution buffer to give a final activity concentration of 20 mM min-1; prepare daily Pyruvate solution: 40 mM pyruvate, sodium salt (Merck), freshly prepared Solutions of KIC, NADH, PLP, 2-OG, and enzyme dilution buffer: Prepare solutions as described above for the BAM assay procedure Check of Coupling Enzyme Activities. The coupling enzyme working solutions may be checked as follows. Dilute the working solutions 1 : 100 with enzyme dissolution buffer. Enzyme activities are then assessed in a 10-mm cuvette at 25° by measuring spectrophotometrically the NADH consumption at 334 nm against time. The assays comprise (1-ml final volume) the following: for ASAT (specifications for ALAT in parentheses): 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of KIC, NADH, PLP, 2-OG, and MDH (LDH) working solution, and 0.2 ml of 1--I20.The reaction is started by addition of 0.05 ml of ASAT (ALAT); for MDH (specifications for LDH in parentheses): 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of KIC, NADH, PLP, and OAA (pyruvate) solution, and 0.25 ml of H20. The reaction is started by addition of 0.05 ml of diluted MDD (LDH) solution. Under these conditions, the diluted ASAT (ALAT) preparation should yield a linear decrease in absorbance of 0.031 AA min -1, and the diluted MDH (LDH) preparation should yield an absorbance change of 0.062 &A min -~ (___-10%).
30
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
Branched-Chain L-Amino-Acid Aminotransferase Measurement Procedure. Enzyme activity is assessed at 25° in a 10-ram cuvette and a reaction volume of 1 ml by measuring spectrophotometrically the linear decrease in NADH absorption at 334 nm against time. For the BAM assay procedure (specifications for the BAL assay in parentheses), the cuvette contains 0.5 ml of BAM buffer (BAL buffer), 0.05 ml each of NADH, PLP, ASAT (ALAT), and MDH (LDH) working solution, and up to 0.25 ml of a (crude) BCAA-AT preparation. When necessary, an appropriate volume of H20 is added to give 0.95 ml. After recording the baseline, the reaction is started by addition of 0.05 ml of KIC solution. When the linear decrease of absorbance exceeds 0.15 AA rain -1, it is advisable to reduce the sample volume or to dilute the BCAA-AT preparation appropriately. For the preparation of additional reagent blanks, the BCAA-AT in the assay is replaced by an appropriate volume of H20. The latter determination is essential in the case of the BAL assay procedure, because 4-methyl-2-oxopentanoate is also a (although poor) substrate for L-lactate dehydrogenase. After correction of the measured linear change of absorbance per minute for baseline and reagent blank, the BCAA-AT activity concentration of the preparation is calculated by the formula given above.
Performance of Methods Precision and Accuracy. When stoichiometry was checked in the BAM assay, the rates of NADH consumption and L-leucine formation were found to be equivalent. When comparative BCAA-AT activity measurements were performed with the BAM and BAL assays, practically identical results were obtained (linear regression analysis: y = 0.41 + 0.93x, r = 0.998, n = 10). In addition, a good linear correlation was found between the BAM assay and a procedure using e-leucine as substrate and o-2-hydroxyisocaproate dehydrogenase as a coupling enzyme (see above). 12 As already noted, the sensitivity of the spectrophotometric assay methods for detection of BCAA-AT activity is better than 0.1/zM min -1. The assays are linear up to a BCAA-AT activity of about 25 ~M min -1. When determined with rat heart BCAA-AT activities of 4.5, 9.7, and 23.0/~M rain -1 in the BAM assays, the coefficient of variation (within run) amounted to 2.3, 1.0, and 1.4%, respectively (n = 5). 12 Temperature Conversion Factors. When related to the rates obtained with partially purified BCAA-AT preparations from rat heart in the BAM assay at 25°, estimated temperature conversion factors for 30 and 37° were 1.4 and 2.3, respectively (n = 6)) z
[3]
ANALYSIS OF B C A A AMINOTRANSFERASE
31
Comments. The present assays are not restricted to 4-methyl-2-oxopentanoate as substrate. Other branched-chain 2-oxo acids as well as straightchain 2-oxo acids can be used as well. Thus, the assays are of general usefulness when studies of the substrate properties of BCAA-AT (iso)enzymes are to be conducted. 12 The present assay conditions have been optimized for analysis of rat heart mitochondrial BCAA-AT. According to the available kinetic data, the reaction rate should amount to 0.9 Vm~,for this enzyme. Some modifications may be needed when the test conditions are to be optimized for analysis of (iso)enzymes from other sources. As with the other NADH-dependent assay procedure using L-leucine as substrate (described above), NH4 + may cause interferences in the BAM and BAL assay procedures and must be omitted from the assay.
Sample Preparation Procedures for the extraction and purification of branched-chain Lamino-acid aminotransferase activity have been described (see Refs. 16 and 17). When the procedure described here is to be used in crude tissue extracts, laboratory animals should be heparinized and the organs thoroughly rinsed, e.g., by perfusion with ice-cold saline (0.154 M NaC1), prior to extraction in order to remove interfering hemoglobin. With rat hearts, the following extraction procedure is applied: After rinsing by retrograde perfusion, the blood-free organ is frozen with liquid N2 and pulverized in a percussion mortar, and the powder is extracted by treatment with a 10-fold volume (w/v) of potassium phosphate buffer (200 mM, pH 6.0, 5 mM EDTA) and centrifugation. For extraction of bovine aorta endothelial cells, microcarrier cultures at confluency are washed three times with a twofold volume of ice-cold phosphate-buffered saline, and cell-free extracts are then prepared by addition of an equal volume of extraction buffer [50 mM Tris-HC1 (pH 7.4), 2mM EDTA, 0.2% (v/v) Triton X-100] and centrifugation. 11'12The latter extraction procedure has also been applied to cultured human skin fibroblasts. Tissue Activities and Kinetic Properties Branched-chain L-amino-acid aminotransferase activities have been reported for a number of tissues from various mammalian species 1-4 and for 16Z. K. Korpela, Methods Enzymol. 166, 269 (1988). 17 R. Kido, Methods Enzymol. 166, 275 (1988).
32
SUBSTRATE PREPARATION, ENZYME ASSAYS, INHIBITORS
[3]
One study compared enzyme activities in rat, human, and monkey tissue and found considerable tissue and species differences. 6 Because numerous, quite different assay conditions have been applied it is hardly possible, however, to compare the enzyme activity data obtained in different laboratories (see Refs. 12 and 18 for discussion and references). The human mitochondrial (iso)enzyme, quite comparable to enzymes from other mammalian sources, exhibits a rather broad substrate specificity. 1'a8 With L-leucine (1 mM), the activity against amino group acceptors (forward reaction) was in the order 2-oxoglutarate -> branched-chain > straight-chain 2-oxo acids (C3-C8). With 4-methyl-2-oxopentanoate (1 mM) the activity against amino group donors (reverse reaction) was in the order L-glutamate ~ branched-chain > straight chain (C3-C6) > other L-amino acids) 8 With respect to the kinetic properties of the enzyme, systematic studies using adequate methodology 19 appear not to have been published. Our studies of the Michaelis constants of the rat heart mitochondrial (iso)enzyme yielded the following results (Km,app., means ___ asymptotic standard errors): with u-leucine and 2-oxoglutarate as substrates, 0.30 ___ 0.02 and 0.68 _ 0.03 mM, respectively; with branched-chain 2-oxo acid substrates and L-glutamate (Kin,app. for the amino group donor in parentheses): 4-methyl-2-oxopentanoate (KIC), 0.14 _+ 0.01 (6.65 ___ 0.34) mM; 3methyl-2-oxobutanoate (KIV), 0.11 _ 0.01 (3.60 --- 0.28) mM; (R,S)-3methyl-2-oxopentanoate [(R,S)-KMV], 0.07 _ 0.01 (2.45 _ 0.19); Ki,app. values amounted to 2.10 --- 0.18, 4.19 _ 0.74, and 1.57 --- 0.21 mM, respectively. H,12 Using cell-free extracts from cultured fibroblasts, the following Km,app. values were found for the human mitochondrial (iso)enzyme2°: forward reaction with 2-oxoglutarate (Km,~pp. for the common amino group acceptor in parentheses): e-Leu, 0.62 _+ 0.04 (3.8 --+ 0.2) mM; u-Val, 2.96 -- 0.11 (4.0 _ 0.1) mM; e-Ile, 0.56 ___ 0.03 (5.5 ___ 0.2) mM; L-alloisoleucine, 1.54--- 0.07 (2.4 ---+ 0.1) mM. For 2-oxoglutarate, the estimated Ki,~pp was >60 mM. For the reverse reaction with eglutamate (Km,app for the common amino group donor in parentheses) Kin,app. estimates were as follows: KIC, 0.53 ___ 0.03 (28.3 _-_ 1.6) mM; KIV, 0.61 --- 0.03 (10.4 ___ 0.5) mM; (S)-KMV, 0.17 _ 0.02 (21.3 --- 2.3) mM; (R)-KMV, 0.06 _+ 0.01 (4.5 --- 0.5) mM. Estimated Ki,~pp. values with branched-chain 2-oxo acid substrates were ->-5 mM. No inhibitory effects of L-amino acids were noted. c u l t u r e d cells. 12,x8
18p. Schadewaldt, U. Wendel, and H.-W. Hammen, Amino Acids 9, 147 (1995). 19 R. D. Allison and D. L. Purich, Methods Enzymol. 63, 3 (1979). 20 p. Schadewaldt and H.-W. Hammen, unpublished data, 1999.