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Beef heart H4 lactate dehydrogenase: N-terminal and C-terminal residues
BIOCHIMICAET BIOPHYSICAACTA
31
BBA 35929 B E E F H E A R T H 4 LACTATE D E H Y D R O G E N A S E : N - T E R M I N A L AND C-TERMINAL R E S I D U E S
L E W I S D. STEGINK, BARBARA M. SANBORN, MARVIN C. BRUMMEL AND CARL S. VESTLING
Departments of Pediatrics and Biochemistry, The University of Iowa College of Medicine, Iowa City, Iowa 52240 (U.S.A.) (Received April 26th, 1971)
SUMMARY
N-terminal analyses of beef heart H 4 lactate dehydrogenase involving the use of fluorodinitrobenzene, phenylisothiocyanate, or 2,4,6-trinitrobenzene sulfonic acid failed to demonstrate the presence of a free amino acid at the amino terminus of the protein in contrast to an earlier report (E. APPELA AND R. ZlTO, Ann. N . Y . Acad. Sci., 151 (1968) 568). Quantitative determination of bound acetyl groups on several batches of enzyme gave values of 3.2-4.6 moles/mole of protein (molecular weight 131 ooo). The N-terminal peptide, characterized as N-acetylalanyl-threonine, was isolated from pronase digests of the protein, and quantitative recovery experiments using 3H-labeled N-acetylalanylthreonine as an internal standard showed that all of the acetyl residues in commercial preparations are found at the amino termini. Leucine was demonstrated to be the C-terminal amino acid in the H 4 isozyme using a tritium exchange method while both leucine and phenylalanine were present at the C-terminus in a mixture of H, and H3M isozymes.
INTRODUCTION
APPELLAAND ZITO1 reported that while bovine heart H a lactate dehydrogenase failed to react with either fluorodinitrobenzene or phenylisothiocyanate under the usual N-terminal analysis conditions, 8 N-terminal valine residues were found when the 2,4,6-trinitrobenzene sulfonic acid method of OKUYANA AND SATAKE2 was employed. These data were consistent with earlier data reported by APPELLA3 suggesting the presence of 8 polypeptide chains per mole of this enzyme. These authors postulated that the 2,4,6-trinitrobenzene sulfonic acid method might lead in some way to the hydrolysis of the N-acetyl groups reported to be present on this isozyme by STEGINK AND VESTLING 4, accounting for the difference in reactivity of the enzyme with the various N-terminal reagents. In our experiments, bovine heart H a lactate dehydrogenase failed to yield stoichiometric quantities of free N-terminal amino acids when analyzed by either Biochim. Biophys. Acta, 251 (197 I) 31-37
32
L.D. STEGINK et al.
the fluorodinitrobenzene, phenylisothiocyanate, or 2,4,6-trinitrobenzene sulfonic acid methods. These findings, and our earlier demonstration of the presence of stoichiometric quantities of N-acetyl groups in this protein 4 strongly suggested the presence of an acetylated N-terminal amino acid. This paper reports the isolation and characterization of the N-terminal peptide (N-acetylalanylthreonine), and confirms the presence of leucine as the C-terminal residue.
MATERIALS AND METHODS
Beef heart lactate dehydrogenase (either Worthington, or Sigma Type III), dialyzed against 5 mM potassium phosphate buffer (pH 6.5) containing i mM 2mercaptoethanol, was purified by chromatography on a DEAE-cellulose column (Schleicher and Schuell, 2. 5 cm × 4o cm) equilibrated with the same buffer 5. The lactate dehydrogenase isozymes were eluted with a linear gradient of o-o.o2 M NaC1 in the same buffer, and the fractions corresponding to the H3M and H 4 isozymes pooled, concentrated in an Amicon ultrafiltration apparatus and frozen. Starch-gel electrophoresis using the method of FI~E AND COST~LLO6 indicated that the H 4 isozyme fraction was free of other isozymes, although the HaM fraction contained significant amounts of the H 4 isozyme. Acrylamide gel electrophoresis (7.5% gel, p H 8.5) of the H 4 isozyme showed a single, sharp, intense band when stained either for lactate dehydrogenase activity or protein. The molecular weight of the isozyme was assumed to be I 3 I ooo (refs. 7, 8). Pronase (Calbiochem, Grade B, 45 ooo PUK/g) was passed through a column of Sephadex G-25 to remove free acetate. Pronase used for the quantitative experiments was free of acetate. N-terminal analyses using fluorodinitrobenzene and phenylisothiocyanate were carried out as described earlier for rat liver lactate dehydrogenase 4, and those using 2,4,6-trinitrobenzene sulfonic acid as described by OKUYANA AND SATAKE2. The C-terminal residues of the isolated peptides were determined by Method C of HOLCOMB et al2, using aH20 (New England Nuclear, I C/g). After acid hydrolysis (6 M HC1, 19 h, IIO°), the mixture was spotted on a sheet of W h a t m a n No. I paper (48 cm × 57 cm) along with 5 #1 of a standard amino acid mixture (Technicon Instruments, 2. 5/~mole of each amino acid per ml), and separated by two-dimensional chromatography in (a) isopropanol-formic acid-water (4o:2:IO, b y vol.) and (b) nbutanol-acetone-diethylamine-water (IO :IO :2:5, b y vol.). The spots were detected by ninhydfin spray, cut out, placed in IO ml of BRAY'S1° solution and counted in a Packard TriCarb spectrometer. C-terminal analyses of the intact lactate dehydrogenase were carried out by a modification of the method of HOLCOMB et al. 9 as previously described n using 2-4 mg protein. The final residue, after acid hydrolysis, was dissolved in I.O ml of 3.5% sulfosalicylic acid and placed on a Technicon NC-I single column amino acid analyzer equipped with a flow cell assembly for simultaneous radioactivity and amino acid analysis. The column effluent was passed directly into a i-ml anthracene-packed flow cell (Nuclear Chicago) placed in a Unilux I I A (Nuclear Chicago) scintillation counter prior to mixing with ninhydrin. In this way, both amino acid analysis and the quantity of radioactivity in each amino acid were determined simultaneously, eliminating Biochim. Biophys. Acta, 251 (1971) 31-37
N- AND C-TERMINALRESIDUES
33
the necessity for two-dimensional paper chromatography. The buffer system described by EFRON1~ was used for the amino acid analyzer. N-Acetyl-L-threonyl-L-alanine and N-acetyl-L-alanyl-L-threonine were synthesized and purified as previously described u. N-E3H]acetyl-L-alanyl-L-threonine was synthesized using [3Hlacetic anhydride (New England Nuclear, 50 mC/mmole). High-voltage electrophoresis was performed in 3.3~/o acetic acid titrated to p H 3.6 with pyridine (26 V/cm, Whatman No. I, 2 h). Amino acids were detected with ninhydrin spray, and N-acetylated peptides were detected by the method of RYDON AND SMITH1~. Acetyl group determinations were carried out by the method of STEGINK 14.
The N-terminal peptide was isolated from heat denatured lactate dehydrogenase following the basic technique we have described earlier 15. The lactate dehydrogenase (300 mg) was digested with 6 mg of pronase added in 3 equal aliquots over the course of 22 h (pH 7.8, 37 °, I mM CaC12, ioo ml). The digest was acidified to pH 3, centrifuged to remove insoluble matter, passed over a AG5oW-X2 (H +) column (Biorad, 200-400 mesh, 3.5 cm X 46 cm), and the effluent (400 ml) concentrated by lyophilization. The residue was dissolved in water, the pH adjusted to 6. 7, and chromatographed on a Rexyn 2Ol (C1-) column (Fisher, 200-400 mesh, I cm X 58 cm) with a linear gradient of 14o ml of water and 14o ml of 0.02 M HCI. The effluent was collected in 2-ml fractions, pooled according to absorbance at 215 nm and the individual pooled fractions lyophilized. The residue was dissolved in a small amount of water in each case, assayed for the presence of acetyl groups, and analyzed for amino acid content after hydrolysis for 15 h in constant boiling HC1 at IiO °. Quantitative estimates of the terminal peptide were performed on 300 mg of the H4 lactate dehydrogenase by adding E~H]acetyl-L-alanyl-L-threonine (IO #C/#mole) to the enzyme prior to pronase digestion and following the recovery of the radioactivity and total acetyl groups during the isolation procedure. RESULTS
Triplicate analyses of each of the two purified H 4 beef heart lactate dehydrogenase preparations studied indicated the presence of 3.2-4.6 moles of acetate per molecular weight of 131 ooo. N-terminal analyses using fluorodinitrobenzene, phenylisothiocyanate or 2,4,6-trinitrobenzene sulfonic acid failed to detect molar quantities of any N-terminal amino acid. APPELLA AND ZITO1 reported finding 8 N-terminal valine residues in beef heart H 4 lactate dehydrogenase using the 2,4,6-trinitrobenzene sulfonic acid method. They postulated that the conditions of the method might lead in some way to the hydrolysis of the N-acetyl groups previously noted on this enzyme 4, accounting for the differences noted between this method and the fluorodinitrobenzene and phenylisothiocyanate methods which failed to yield stoichiometric quantities of free N-terminal amino acids 1. In an attempt to investigate this possibility, the 2,4,6-trinitrobenzene sulfonic acid method was studied in greater detail using bovine trypsinogen, which has an N-terminal valine residue 16, as a model. Because of large losses (50% or greater) of trinitrophenyl derivatives occurring during the acid hydrolysis step, it was necessary to adjust reaction conditions to obtain maximal release of trinitrophenylvaline from the protein with minimal decomposition of the compound. Hydrolysis of triBiochim. Biophys. Acta, 251 (1971) 31-37
34 I
I
I
I
L. D, S T E G I N K et
al.
2Ol (C1-) c o l u m n . T h e l i n e a r g r a d i e n t
of
I
I
100
120
m 1.0
~Eae (M v
I
wO.(~
o
if) rn
< 02
20
40 60 80 FRACTION NUMBER
F i g . I. T h e e l u t i o n p a t t e r n o f p e p t i d e s f r o m a R e x y n w a t e r - o . o 2 M HC1 w a s s t a r t e d a t t h e a r r o w .
nitrophenylated trypsinogen for 4 h yielded o.13 mole trinitrophenylvaline per mole protein after correction for 49% recovery of standard, while a 7.5-h hydrolysis yielded 0.73 mole trinitrophenylvaline per mole protein after correction for 12% recovery of added standard. Assay of beef heart H 4 and rat liver M4 lactate dehydrogenase under the latter conditions yielded submolar quantities of a variety of trinitrophenylated amino acids, which were similar in both quantity and type to those reported in our earlier studies of the rat liver enzyme using other methods 4. No evidence was obtained indicating the presence of 4 or 8 N-terminal valine residues, hydrolysis of protein bound acetyl residues or deacetylation of standard N-acetylvaline by this method. The acetylated N-terminal peptide was isolated using the technique first described by NARITA1L The pronase digest was acidified, passed through a AG5oW-X2 column and chromatographed on a Rexyn 2Ol (C]-) column resolving the mixture into 4 peaks (Fig. z). Fraction II1 was ninhydrin negative, contained 73% of the acetate recovered from the Rexyn 2Ol column, and equimolar quantities of acetate, alanine and threonine as well as minor quantities of other amino acids after acid hydrolysis (Table I). C-terminal analysis of the isolated peptide using the tritium exchange technique demonstrated that threonine was the C-terminal amino acid. TABLE
I
AMINO ACID AND ACETYL GROUP COMPOSITION AFTER
DIGESTION
OF
BEEF
HEART
OF FRACTION
H 4 LACTATE
Compound
]*mole
Amino acid/acetate
Aspartate Threonine Serine Glutamate Glycine Alanine Acetate
o.o21 o. 144 0.009 0.008 0.009 o. 15 ° o. 148
o.i 4 0.98 0.06 0.06 0.06 I .oi --
Biochim. Biophys. Acta, 251 ( I 9 7 1 ) 3 1 - 3 7
III
ELUTED
DEHYDROGENASE
FROM
WITH
REXYN
PRONASE
2OI
COLUMN
N- AND C-TERMINALRESIDUES
35
Fig. 2. High-voltage paper electrophoresis of N-acetylalanylthreonine, N-acetylthreonylalanine, and the isolated N-terminal peptide from beef heart lactate dehydrogenase before and after acid hydrolysis (I M HC1, 5 h, 6o°): N-acetylthreonylalanine, before (A) and after (B) hydrolysis; the N-terminal lactate dehydrogenase peptide, before (C) and after (D) hydrolysis; and N-acetylalanylthreonine, before (E) and after (F) hydrolysis. ALLISON et al. TM reported the isolation of N-acylthreonylalanylleucine (acyl group not identified) from subtilisin digests of dogfish muscle M 4 lactate dehydrogenase. They reported that the acyl group of this peptide was acid labile, and was removed b y mild acid hydrolysis (I M HC1, 37 °, 5 h). In our experiments, synthetic N-acetylthreonylalanine was substantially, but not completely deacetylated under these conditions, while N-acetylalanylthreonine and the isolated peptide were essentially unchanged as judged both by reactivity with ninhydrin and behavior on high voltage electrophoresis. As shown in Fig. 2, complete deacetylation of the Nacetylthreonylalanine was obtained in our experiments at 60 ° (I M HC1, 5 h), while N-acetylalanylthreonine and the isolated peptide were hydrolyzed about 30% under these conditions. The acid hydrolyzed N-acetylthreonylalanine spot was deliberately overloaded to demonstrate the complete deacetylation of this peptide. N-Acetylalanylthreonine separates slightly from N-acetylthreonylalanine in this system after electrophoresis for 3 h, and the isolated peptide migrates at the same rate as does the N-acetylalanylthreonine. Since the microenzymatic assay is specific for acetate 14, and since we have confirmed the identity of the acyl group in rat liver M4 lactate dehydrogenase as acetate b y a separate method 15, the identity of the blocking group is most certainly acetate. Thus, the structure of the N-terminal peptide is N-acetylalanylthreonine. The acetylated peptide was also isolated from a batch of beef h e a r t H 4 lactate dehydrogenase (192 mg) to which [aHJacetylalanylthreonine had been added during pronase digestion to determine what portion of the acetyl groups present on the original enzyme were recovered as this peptide. After correction for the loss of the labeled internal standard peptide during the isolation procedure, 90°/0 of the original protein bound acetyl groups could be accounted for as N-acetylalanylthreonine. C-terminal analyses of intact beef heart H 4 lactate dehydrogenase using the tritium exchange method demonstrated that leucine is the C-terminal amino acid Biochim. Biophys. Acta, 251 (1971) 31-37
36
L.D. STEGINK et al.
TABLE II C-TERMINAL TRITIUM EXCHANGE ANALYSES OF INTACT AND H3~ LACTATE DEttYDROGENASE ISOZYMES
Amino acid
HEART
H4 A N D
A MIXTURE
OF H 4
Counts/4 rain* Ha (3.6rag)
Aspartate Threonine Glutamate Leucine Phenylalanine Lysine Histidine
BEEF
655 322 8385 80 IIo 525
H a + H3M (2.2rag)
25o 7° 25o 2650 28o0 45°
* Not quench corrected. in this isozyme as shown in Table I I while the mixture of the H 4 and H3M isozymes contained both leucine and phenylalanine as C-terminal amino acids. Only minor quantities of radioactivity were noted in other amino acids. DISCUSSION We have previously reported the presence of stoichiometric quantities of Nacetyl groups in beef heart H 4 lactate dehydrogenase suggesting the presence of Nacetylated amino acids at the N-terminal. APPELLA AND ZITO1 reported the failure of the beef heart H 4 enzyme to react with fluorodinitrobenzene and phenylisothiocyanate under the usual N-terminal conditions, but reported that 8 N-terminal valine residues were obtained with the 2,4,6-trinitrobenzene method: They suggested that the difference between these findings could be explained if the latter method led in some way to the hydrolysis of the N-terminal acetyl group. We have been unable to demonstrate the presence of stoichiometric quantities of free N-terminal amino acids in the beef heart H 4 lactate dehydrogenase using either fluorodinitrobenzene, phenylisothiocyanate or 2,4,6-trinitrobenzene sulfonic acid methods. Studies with N-acetylated amino acids and peptides failed to detect substantial hydrolysis of the acetyl groups during reaction with 2,4,6-trinitrobenzene sulfonic acid. Subsequently, we isolated the N-terminal peptide from this enzyme, characterizing it as N-acetylalanylthreonine. Thus, we are unable to explain the reports of APPELLA AND ZITO1. If some modification of the sample of enzyme studied by these authors had occurred, it did not represent a simple deacetylation of the N-terminal amino acid residue or removal of the N-acetyl N-terminal amino acid, since valine was not found at either of these positions. C-terminal analyses of the beef heart H 4 isozyme using the tritium exchange method confirmed the presence of leucine at the C-terminus of the enzyme, in agreement with the carboxypeptidase and hydrazinolysis data reported by APPELLA AND ZITO1. Thus, leucine is the C-terminal amino acid of the H-type lactate dehydrogenase subunits in the chicken heart H 4 (ref. II), pig heart H 4 (ref. I9), and beef heart H4 isozymes, while phenylalanine is the C-terminal residue in the M-type subunits found Biochim. Biophys. Acta, 251 (1971) 31-37
N-
AND C-TERMINAL RESIDUES
37
in rat liver M4 (ref. 20), dogfish muscle M4 (ref. 18), pig muscle M a (reL 19) and rabbit muscle M, (ref. 21) isozymes. As expected from the above data, C-terminal leucine and phenylalanine were found in the H3M beef heart isozyme. The data presented demonstrate that this method cannot be used to quantitate C-terminal residues, since nearly equal amounts of tritium were incorporated into leucine and phenylalanine, whereas the expected ratio would be 3 leucine per phenylalanine. The N-terminal residues of the M and H subunits of lactate dehydrogenase also differ. We have shown that rat liver M4 (ref. 15) and rabbit muscle M a (ref. 21) isozymes have N-terminal N-acetylalanylalanine sequences, while chicken heart H4 (ref. II) and beef heart isozymes have N-terminal N-acteylalanylthreonine sequences. ALLISON et al. is have reported that dogfish muscle M4 lactate dehydrogenase contains an N-acylthreony|alanylleucine N-terminal sequence. However, the C-terminal tritium exchange data and demonstrated acid stability of the isolated peptide, as well as the demonstrated acid lability of N-acetylthreonylalanine substantiate the contention that the N-terminal peptide found in the beef heart H 4 enzyme is N-acetylalanylthreonine. Thus, the N-terminal sequence reported for the M subunit of the dogfish differs both from the N-terminal sequence of the M subunits from rat and rabbit and from the N-terminal sequence of the bovine and avian H subunits studied. ACKNOWLEDGMENTS
This collaborative effort was supported by the Mary F. Novak Memorial Grant for Cancer Research P-538, American Cancer Society (L.D.S.) and Grant CA 07617-07, National Institutes of Health (C.S.V.). REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21
E. APPELLA AND R. ZITO, Ann. N . Y . Acad. Sci., 151 (1968) 568. T. OKUYAMA AND K. SATAKE, J. Bioehem. Tokyo, 47 (196o) 454. E. APPELLA, Brookhaven Syrup. Biol., 17 (1964) 151. L. D. STEGINK AND C. S. VESTLING, J. Biol. Chem., 241 (1966) 4923. A. PESCE, T. P. FONDY, F. STOLZENBACH, F. CASTILLO AND N. O. KAPLAN, J. Biol. Chem., 242 (1967) 2151. I. H. FINE AND L. A. COSTELLO, Methods Enzymol., 6 (1963) 958. C. A. MARKERT AND E. APPELLA, Ann. N . Y . Aead. Sci., 94 (1961) 678. A. PESCE, R. H. MCKAY, F. STOLZ]~NBACH, R. D. CAHN AND N. O. KAPLAN, jr. Biol. Chem., 239 (1964) 1753. G. N. HOLCOMB, S. A. JAMES AND D. N. WARD, Biochemistry, 7 (1968) 1291. G. A. BRAY, Anal. Biochem., i (196o) 27. M. C. BRUMMEL, ]3. M. SANBORN AND L. D. STEGINK, Arch. Bioehem. Biophys., 143 (1971) 33 o. M. L. E~RON, in L. K. SKEGGS, JR., Automation in Analytical Chemistry, Mediad Incorp., New York, New York, 1966, p. 637. H. N. RYDON AND P. W. G. SMITH, Nature, 169 (I952) 922. L. D. STEGINK, Anal. Biochem., 20 (1967) 502. B. M. SANBORN, M. C. BRUMMEL, L. D. STEGINK AND C. S. VESTLING, Biochim. Biophys. Acta, 221 (197 ° ) 125. M. ROVERY, C. FABRE AND P. DESNUELLE, Biochim. Biophys. Acta, 9 (1952) 702. K. NARITA, Biochim. Biophys. Acta, 28 (1958) 184. W. S. ALLISON, J. ADMIRAAL AND N. O. KAPLAN, J. Biol. Chem., 244 (1968) 4743. K. MELLA, H. J. TORFF, T. J. F6LSCHE AND G. PFLEIDERER, Z. Physiol. Chem., 35 ° (1968) 28. W. T. HSlEH, L. E. GUNDERSEN AND C. S. VESTLING, Biochem. Biophys. Res. Commun., 43 (1971) 69. M. C. BRUMMEL AND L. D. STEGINK, Comp. Biochem. Physiol., submitted for publication.
Biochim. Biophys. Acta, 251 (1971) 31-37
31
BBA 35929 B E E F H E A R T H 4 LACTATE D E H Y D R O G E N A S E : N - T E R M I N A L AND C-TERMINAL R E S I D U E S
L E W I S D. STEGINK, BARBARA M. SANBORN, MARVIN C. BRUMMEL AND CARL S. VESTLING
Departments of Pediatrics and Biochemistry, The University of Iowa College of Medicine, Iowa City, Iowa 52240 (U.S.A.) (Received April 26th, 1971)
SUMMARY
N-terminal analyses of beef heart H 4 lactate dehydrogenase involving the use of fluorodinitrobenzene, phenylisothiocyanate, or 2,4,6-trinitrobenzene sulfonic acid failed to demonstrate the presence of a free amino acid at the amino terminus of the protein in contrast to an earlier report (E. APPELA AND R. ZlTO, Ann. N . Y . Acad. Sci., 151 (1968) 568). Quantitative determination of bound acetyl groups on several batches of enzyme gave values of 3.2-4.6 moles/mole of protein (molecular weight 131 ooo). The N-terminal peptide, characterized as N-acetylalanyl-threonine, was isolated from pronase digests of the protein, and quantitative recovery experiments using 3H-labeled N-acetylalanylthreonine as an internal standard showed that all of the acetyl residues in commercial preparations are found at the amino termini. Leucine was demonstrated to be the C-terminal amino acid in the H 4 isozyme using a tritium exchange method while both leucine and phenylalanine were present at the C-terminus in a mixture of H, and H3M isozymes.
INTRODUCTION
APPELLAAND ZITO1 reported that while bovine heart H a lactate dehydrogenase failed to react with either fluorodinitrobenzene or phenylisothiocyanate under the usual N-terminal analysis conditions, 8 N-terminal valine residues were found when the 2,4,6-trinitrobenzene sulfonic acid method of OKUYANA AND SATAKE2 was employed. These data were consistent with earlier data reported by APPELLA3 suggesting the presence of 8 polypeptide chains per mole of this enzyme. These authors postulated that the 2,4,6-trinitrobenzene sulfonic acid method might lead in some way to the hydrolysis of the N-acetyl groups reported to be present on this isozyme by STEGINK AND VESTLING 4, accounting for the difference in reactivity of the enzyme with the various N-terminal reagents. In our experiments, bovine heart H a lactate dehydrogenase failed to yield stoichiometric quantities of free N-terminal amino acids when analyzed by either Biochim. Biophys. Acta, 251 (197 I) 31-37
32
L.D. STEGINK et al.
the fluorodinitrobenzene, phenylisothiocyanate, or 2,4,6-trinitrobenzene sulfonic acid methods. These findings, and our earlier demonstration of the presence of stoichiometric quantities of N-acetyl groups in this protein 4 strongly suggested the presence of an acetylated N-terminal amino acid. This paper reports the isolation and characterization of the N-terminal peptide (N-acetylalanylthreonine), and confirms the presence of leucine as the C-terminal residue.
MATERIALS AND METHODS
Beef heart lactate dehydrogenase (either Worthington, or Sigma Type III), dialyzed against 5 mM potassium phosphate buffer (pH 6.5) containing i mM 2mercaptoethanol, was purified by chromatography on a DEAE-cellulose column (Schleicher and Schuell, 2. 5 cm × 4o cm) equilibrated with the same buffer 5. The lactate dehydrogenase isozymes were eluted with a linear gradient of o-o.o2 M NaC1 in the same buffer, and the fractions corresponding to the H3M and H 4 isozymes pooled, concentrated in an Amicon ultrafiltration apparatus and frozen. Starch-gel electrophoresis using the method of FI~E AND COST~LLO6 indicated that the H 4 isozyme fraction was free of other isozymes, although the HaM fraction contained significant amounts of the H 4 isozyme. Acrylamide gel electrophoresis (7.5% gel, p H 8.5) of the H 4 isozyme showed a single, sharp, intense band when stained either for lactate dehydrogenase activity or protein. The molecular weight of the isozyme was assumed to be I 3 I ooo (refs. 7, 8). Pronase (Calbiochem, Grade B, 45 ooo PUK/g) was passed through a column of Sephadex G-25 to remove free acetate. Pronase used for the quantitative experiments was free of acetate. N-terminal analyses using fluorodinitrobenzene and phenylisothiocyanate were carried out as described earlier for rat liver lactate dehydrogenase 4, and those using 2,4,6-trinitrobenzene sulfonic acid as described by OKUYANA AND SATAKE2. The C-terminal residues of the isolated peptides were determined by Method C of HOLCOMB et al2, using aH20 (New England Nuclear, I C/g). After acid hydrolysis (6 M HC1, 19 h, IIO°), the mixture was spotted on a sheet of W h a t m a n No. I paper (48 cm × 57 cm) along with 5 #1 of a standard amino acid mixture (Technicon Instruments, 2. 5/~mole of each amino acid per ml), and separated by two-dimensional chromatography in (a) isopropanol-formic acid-water (4o:2:IO, b y vol.) and (b) nbutanol-acetone-diethylamine-water (IO :IO :2:5, b y vol.). The spots were detected by ninhydfin spray, cut out, placed in IO ml of BRAY'S1° solution and counted in a Packard TriCarb spectrometer. C-terminal analyses of the intact lactate dehydrogenase were carried out by a modification of the method of HOLCOMB et al. 9 as previously described n using 2-4 mg protein. The final residue, after acid hydrolysis, was dissolved in I.O ml of 3.5% sulfosalicylic acid and placed on a Technicon NC-I single column amino acid analyzer equipped with a flow cell assembly for simultaneous radioactivity and amino acid analysis. The column effluent was passed directly into a i-ml anthracene-packed flow cell (Nuclear Chicago) placed in a Unilux I I A (Nuclear Chicago) scintillation counter prior to mixing with ninhydrin. In this way, both amino acid analysis and the quantity of radioactivity in each amino acid were determined simultaneously, eliminating Biochim. Biophys. Acta, 251 (1971) 31-37
N- AND C-TERMINALRESIDUES
33
the necessity for two-dimensional paper chromatography. The buffer system described by EFRON1~ was used for the amino acid analyzer. N-Acetyl-L-threonyl-L-alanine and N-acetyl-L-alanyl-L-threonine were synthesized and purified as previously described u. N-E3H]acetyl-L-alanyl-L-threonine was synthesized using [3Hlacetic anhydride (New England Nuclear, 50 mC/mmole). High-voltage electrophoresis was performed in 3.3~/o acetic acid titrated to p H 3.6 with pyridine (26 V/cm, Whatman No. I, 2 h). Amino acids were detected with ninhydrin spray, and N-acetylated peptides were detected by the method of RYDON AND SMITH1~. Acetyl group determinations were carried out by the method of STEGINK 14.
The N-terminal peptide was isolated from heat denatured lactate dehydrogenase following the basic technique we have described earlier 15. The lactate dehydrogenase (300 mg) was digested with 6 mg of pronase added in 3 equal aliquots over the course of 22 h (pH 7.8, 37 °, I mM CaC12, ioo ml). The digest was acidified to pH 3, centrifuged to remove insoluble matter, passed over a AG5oW-X2 (H +) column (Biorad, 200-400 mesh, 3.5 cm X 46 cm), and the effluent (400 ml) concentrated by lyophilization. The residue was dissolved in water, the pH adjusted to 6. 7, and chromatographed on a Rexyn 2Ol (C1-) column (Fisher, 200-400 mesh, I cm X 58 cm) with a linear gradient of 14o ml of water and 14o ml of 0.02 M HCI. The effluent was collected in 2-ml fractions, pooled according to absorbance at 215 nm and the individual pooled fractions lyophilized. The residue was dissolved in a small amount of water in each case, assayed for the presence of acetyl groups, and analyzed for amino acid content after hydrolysis for 15 h in constant boiling HC1 at IiO °. Quantitative estimates of the terminal peptide were performed on 300 mg of the H4 lactate dehydrogenase by adding E~H]acetyl-L-alanyl-L-threonine (IO #C/#mole) to the enzyme prior to pronase digestion and following the recovery of the radioactivity and total acetyl groups during the isolation procedure. RESULTS
Triplicate analyses of each of the two purified H 4 beef heart lactate dehydrogenase preparations studied indicated the presence of 3.2-4.6 moles of acetate per molecular weight of 131 ooo. N-terminal analyses using fluorodinitrobenzene, phenylisothiocyanate or 2,4,6-trinitrobenzene sulfonic acid failed to detect molar quantities of any N-terminal amino acid. APPELLA AND ZITO1 reported finding 8 N-terminal valine residues in beef heart H 4 lactate dehydrogenase using the 2,4,6-trinitrobenzene sulfonic acid method. They postulated that the conditions of the method might lead in some way to the hydrolysis of the N-acetyl groups previously noted on this enzyme 4, accounting for the differences noted between this method and the fluorodinitrobenzene and phenylisothiocyanate methods which failed to yield stoichiometric quantities of free N-terminal amino acids 1. In an attempt to investigate this possibility, the 2,4,6-trinitrobenzene sulfonic acid method was studied in greater detail using bovine trypsinogen, which has an N-terminal valine residue 16, as a model. Because of large losses (50% or greater) of trinitrophenyl derivatives occurring during the acid hydrolysis step, it was necessary to adjust reaction conditions to obtain maximal release of trinitrophenylvaline from the protein with minimal decomposition of the compound. Hydrolysis of triBiochim. Biophys. Acta, 251 (1971) 31-37
34 I
I
I
I
L. D, S T E G I N K et
al.
2Ol (C1-) c o l u m n . T h e l i n e a r g r a d i e n t
of
I
I
100
120
m 1.0
~Eae (M v
I
wO.(~
o
if) rn
< 02
20
40 60 80 FRACTION NUMBER
F i g . I. T h e e l u t i o n p a t t e r n o f p e p t i d e s f r o m a R e x y n w a t e r - o . o 2 M HC1 w a s s t a r t e d a t t h e a r r o w .
nitrophenylated trypsinogen for 4 h yielded o.13 mole trinitrophenylvaline per mole protein after correction for 49% recovery of standard, while a 7.5-h hydrolysis yielded 0.73 mole trinitrophenylvaline per mole protein after correction for 12% recovery of added standard. Assay of beef heart H 4 and rat liver M4 lactate dehydrogenase under the latter conditions yielded submolar quantities of a variety of trinitrophenylated amino acids, which were similar in both quantity and type to those reported in our earlier studies of the rat liver enzyme using other methods 4. No evidence was obtained indicating the presence of 4 or 8 N-terminal valine residues, hydrolysis of protein bound acetyl residues or deacetylation of standard N-acetylvaline by this method. The acetylated N-terminal peptide was isolated using the technique first described by NARITA1L The pronase digest was acidified, passed through a AG5oW-X2 column and chromatographed on a Rexyn 2Ol (C]-) column resolving the mixture into 4 peaks (Fig. z). Fraction II1 was ninhydrin negative, contained 73% of the acetate recovered from the Rexyn 2Ol column, and equimolar quantities of acetate, alanine and threonine as well as minor quantities of other amino acids after acid hydrolysis (Table I). C-terminal analysis of the isolated peptide using the tritium exchange technique demonstrated that threonine was the C-terminal amino acid. TABLE
I
AMINO ACID AND ACETYL GROUP COMPOSITION AFTER
DIGESTION
OF
BEEF
HEART
OF FRACTION
H 4 LACTATE
Compound
]*mole
Amino acid/acetate
Aspartate Threonine Serine Glutamate Glycine Alanine Acetate
o.o21 o. 144 0.009 0.008 0.009 o. 15 ° o. 148
o.i 4 0.98 0.06 0.06 0.06 I .oi --
Biochim. Biophys. Acta, 251 ( I 9 7 1 ) 3 1 - 3 7
III
ELUTED
DEHYDROGENASE
FROM
WITH
REXYN
PRONASE
2OI
COLUMN
N- AND C-TERMINALRESIDUES
35
Fig. 2. High-voltage paper electrophoresis of N-acetylalanylthreonine, N-acetylthreonylalanine, and the isolated N-terminal peptide from beef heart lactate dehydrogenase before and after acid hydrolysis (I M HC1, 5 h, 6o°): N-acetylthreonylalanine, before (A) and after (B) hydrolysis; the N-terminal lactate dehydrogenase peptide, before (C) and after (D) hydrolysis; and N-acetylalanylthreonine, before (E) and after (F) hydrolysis. ALLISON et al. TM reported the isolation of N-acylthreonylalanylleucine (acyl group not identified) from subtilisin digests of dogfish muscle M 4 lactate dehydrogenase. They reported that the acyl group of this peptide was acid labile, and was removed b y mild acid hydrolysis (I M HC1, 37 °, 5 h). In our experiments, synthetic N-acetylthreonylalanine was substantially, but not completely deacetylated under these conditions, while N-acetylalanylthreonine and the isolated peptide were essentially unchanged as judged both by reactivity with ninhydrin and behavior on high voltage electrophoresis. As shown in Fig. 2, complete deacetylation of the Nacetylthreonylalanine was obtained in our experiments at 60 ° (I M HC1, 5 h), while N-acetylalanylthreonine and the isolated peptide were hydrolyzed about 30% under these conditions. The acid hydrolyzed N-acetylthreonylalanine spot was deliberately overloaded to demonstrate the complete deacetylation of this peptide. N-Acetylalanylthreonine separates slightly from N-acetylthreonylalanine in this system after electrophoresis for 3 h, and the isolated peptide migrates at the same rate as does the N-acetylalanylthreonine. Since the microenzymatic assay is specific for acetate 14, and since we have confirmed the identity of the acyl group in rat liver M4 lactate dehydrogenase as acetate b y a separate method 15, the identity of the blocking group is most certainly acetate. Thus, the structure of the N-terminal peptide is N-acetylalanylthreonine. The acetylated peptide was also isolated from a batch of beef h e a r t H 4 lactate dehydrogenase (192 mg) to which [aHJacetylalanylthreonine had been added during pronase digestion to determine what portion of the acetyl groups present on the original enzyme were recovered as this peptide. After correction for the loss of the labeled internal standard peptide during the isolation procedure, 90°/0 of the original protein bound acetyl groups could be accounted for as N-acetylalanylthreonine. C-terminal analyses of intact beef heart H 4 lactate dehydrogenase using the tritium exchange method demonstrated that leucine is the C-terminal amino acid Biochim. Biophys. Acta, 251 (1971) 31-37
36
L.D. STEGINK et al.
TABLE II C-TERMINAL TRITIUM EXCHANGE ANALYSES OF INTACT AND H3~ LACTATE DEttYDROGENASE ISOZYMES
Amino acid
HEART
H4 A N D
A MIXTURE
OF H 4
Counts/4 rain* Ha (3.6rag)
Aspartate Threonine Glutamate Leucine Phenylalanine Lysine Histidine
BEEF
655 322 8385 80 IIo 525
H a + H3M (2.2rag)
25o 7° 25o 2650 28o0 45°
* Not quench corrected. in this isozyme as shown in Table I I while the mixture of the H 4 and H3M isozymes contained both leucine and phenylalanine as C-terminal amino acids. Only minor quantities of radioactivity were noted in other amino acids. DISCUSSION We have previously reported the presence of stoichiometric quantities of Nacetyl groups in beef heart H 4 lactate dehydrogenase suggesting the presence of Nacetylated amino acids at the N-terminal. APPELLA AND ZITO1 reported the failure of the beef heart H 4 enzyme to react with fluorodinitrobenzene and phenylisothiocyanate under the usual N-terminal conditions, but reported that 8 N-terminal valine residues were obtained with the 2,4,6-trinitrobenzene method: They suggested that the difference between these findings could be explained if the latter method led in some way to the hydrolysis of the N-terminal acetyl group. We have been unable to demonstrate the presence of stoichiometric quantities of free N-terminal amino acids in the beef heart H 4 lactate dehydrogenase using either fluorodinitrobenzene, phenylisothiocyanate or 2,4,6-trinitrobenzene sulfonic acid methods. Studies with N-acetylated amino acids and peptides failed to detect substantial hydrolysis of the acetyl groups during reaction with 2,4,6-trinitrobenzene sulfonic acid. Subsequently, we isolated the N-terminal peptide from this enzyme, characterizing it as N-acetylalanylthreonine. Thus, we are unable to explain the reports of APPELLA AND ZITO1. If some modification of the sample of enzyme studied by these authors had occurred, it did not represent a simple deacetylation of the N-terminal amino acid residue or removal of the N-acetyl N-terminal amino acid, since valine was not found at either of these positions. C-terminal analyses of the beef heart H 4 isozyme using the tritium exchange method confirmed the presence of leucine at the C-terminus of the enzyme, in agreement with the carboxypeptidase and hydrazinolysis data reported by APPELLA AND ZITO1. Thus, leucine is the C-terminal amino acid of the H-type lactate dehydrogenase subunits in the chicken heart H 4 (ref. II), pig heart H 4 (ref. I9), and beef heart H4 isozymes, while phenylalanine is the C-terminal residue in the M-type subunits found Biochim. Biophys. Acta, 251 (1971) 31-37
N-
AND C-TERMINAL RESIDUES
37
in rat liver M4 (ref. 20), dogfish muscle M4 (ref. 18), pig muscle M a (reL 19) and rabbit muscle M, (ref. 21) isozymes. As expected from the above data, C-terminal leucine and phenylalanine were found in the H3M beef heart isozyme. The data presented demonstrate that this method cannot be used to quantitate C-terminal residues, since nearly equal amounts of tritium were incorporated into leucine and phenylalanine, whereas the expected ratio would be 3 leucine per phenylalanine. The N-terminal residues of the M and H subunits of lactate dehydrogenase also differ. We have shown that rat liver M4 (ref. 15) and rabbit muscle M a (ref. 21) isozymes have N-terminal N-acetylalanylalanine sequences, while chicken heart H4 (ref. II) and beef heart isozymes have N-terminal N-acteylalanylthreonine sequences. ALLISON et al. is have reported that dogfish muscle M4 lactate dehydrogenase contains an N-acylthreony|alanylleucine N-terminal sequence. However, the C-terminal tritium exchange data and demonstrated acid stability of the isolated peptide, as well as the demonstrated acid lability of N-acetylthreonylalanine substantiate the contention that the N-terminal peptide found in the beef heart H 4 enzyme is N-acetylalanylthreonine. Thus, the N-terminal sequence reported for the M subunit of the dogfish differs both from the N-terminal sequence of the M subunits from rat and rabbit and from the N-terminal sequence of the bovine and avian H subunits studied. ACKNOWLEDGMENTS
This collaborative effort was supported by the Mary F. Novak Memorial Grant for Cancer Research P-538, American Cancer Society (L.D.S.) and Grant CA 07617-07, National Institutes of Health (C.S.V.). REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21
E. APPELLA AND R. ZITO, Ann. N . Y . Acad. Sci., 151 (1968) 568. T. OKUYAMA AND K. SATAKE, J. Bioehem. Tokyo, 47 (196o) 454. E. APPELLA, Brookhaven Syrup. Biol., 17 (1964) 151. L. D. STEGINK AND C. S. VESTLING, J. Biol. Chem., 241 (1966) 4923. A. PESCE, T. P. FONDY, F. STOLZENBACH, F. CASTILLO AND N. O. KAPLAN, J. Biol. Chem., 242 (1967) 2151. I. H. FINE AND L. A. COSTELLO, Methods Enzymol., 6 (1963) 958. C. A. MARKERT AND E. APPELLA, Ann. N . Y . Aead. Sci., 94 (1961) 678. A. PESCE, R. H. MCKAY, F. STOLZ]~NBACH, R. D. CAHN AND N. O. KAPLAN, jr. Biol. Chem., 239 (1964) 1753. G. N. HOLCOMB, S. A. JAMES AND D. N. WARD, Biochemistry, 7 (1968) 1291. G. A. BRAY, Anal. Biochem., i (196o) 27. M. C. BRUMMEL, ]3. M. SANBORN AND L. D. STEGINK, Arch. Bioehem. Biophys., 143 (1971) 33 o. M. L. E~RON, in L. K. SKEGGS, JR., Automation in Analytical Chemistry, Mediad Incorp., New York, New York, 1966, p. 637. H. N. RYDON AND P. W. G. SMITH, Nature, 169 (I952) 922. L. D. STEGINK, Anal. Biochem., 20 (1967) 502. B. M. SANBORN, M. C. BRUMMEL, L. D. STEGINK AND C. S. VESTLING, Biochim. Biophys. Acta, 221 (197 ° ) 125. M. ROVERY, C. FABRE AND P. DESNUELLE, Biochim. Biophys. Acta, 9 (1952) 702. K. NARITA, Biochim. Biophys. Acta, 28 (1958) 184. W. S. ALLISON, J. ADMIRAAL AND N. O. KAPLAN, J. Biol. Chem., 244 (1968) 4743. K. MELLA, H. J. TORFF, T. J. F6LSCHE AND G. PFLEIDERER, Z. Physiol. Chem., 35 ° (1968) 28. W. T. HSlEH, L. E. GUNDERSEN AND C. S. VESTLING, Biochem. Biophys. Res. Commun., 43 (1971) 69. M. C. BRUMMEL AND L. D. STEGINK, Comp. Biochem. Physiol., submitted for publication.
Biochim. Biophys. Acta, 251 (1971) 31-37