128 SHORT COMMUNICATIONS

ALLISON et al. I° have recently reported the isolation of an N-acylthreonyl- alanylleucine peptide (the N-acyl group was not identified) from dogfish muscle M 4 lactate dehydrogenase by similar methods; thus a definite phylogenetic difference occurs in the amino-terminal peptide sequence of the M 4 enzyme. Preliminary studies in these laboratories show that the peptide N-acetylalanylthreonine can be isolated from both beef and chicken heart H 4 enzyme. Further considerations of the acetyl status of lactate dehydrogenase will be forthcoming.

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.).

Department of Biochemistry and Department of Pediatrics, College of Medicine, The University of Iowa, Iowa City, Iowa 52240 ( U . S . A . )

BARBARA M. SANBORN

MARVIN C. BRUMMEL

LEWIS D. STEGINK

CARL S. VESTLING

I L. D. STEGINK AND C. S. VESTLING, J. Biol. Chem., 241 (1966) 4923. 2 W. T. HSlEH AND C. S. VESTLING, Biochem. Prep., I I (1966) 69. 3 Y. P. LEE AND T. TAKAHASHI, Anal. Biochem., 14 (1966) 71. 4 W. R. GRAY AND B. S. HARTLEY, Bioehem. J., 89 (1963) 59P. 5 K. NARITA, Biochim. Biophys. Acla, 28 (1958) 184. 6 L. D. STEGINK, Anal. Biochem., 20 (1967) 502. 7 G. SCHMER ANO G. KREIL, Anal. Bioehem., 29 (1969) 186. 8 M. TROP AND Y. BIRK, Biochem. J., 116 (197 ° ) 19. 9 b2. T. APPELLA AND R. ZITO, Ann. N. Y. Acad. Sci., 151 (1968) 568.

io W. S. ALLISON, J. ADMIRAAL AND N. O. KAPLAN, J. Biol. Chem., 244 (1969) 4743. I I H. N. RYDON AND P. W. G. SMITH, Nalure, 169 (1952) 922. 12 R. STEIGER, Helv. Chim. Acta, 17 (1934) 563.

Received May i9th, 197o

Biochim. Biophys. Aeta, 221 (197 o) 125 I28

BBA 33236

Chromatographic resolution of lysine-rich histones unaffected by phosphatase or ribonuclease treatment

The resolution of lysine-rich histones into species and tissue-specific patterns by ion-exchange chromatography has been reported1, 2. The molecular basis for this resolution appeared to be differences in amino acid sequences. Although the peptide patterns derived from the tryptic digestion of individual subfractions were very similar, there were a modest number of differences, and it was suggested 3 that lysine- rich histones contain an invariant region of primary structure common to all sub- fractions, coupled to a region which varies from one subfraction to the next. In spite of these apparent differences in amino acid sequence it was conceivable that chro- matographic resolution was due to varying degrees of phosphorylation or to the for- mation of RNA-histone complexes.

Biochim. Biophys. Acta, 22I (197 o) t 2 8 - I 3 I

SHORT COMMUNICATIONS 12 9

03

o+

0 . 2

m m 0 1

i _ _

I I ,

i

0 2 ~

I L 0 , _ _ I I 0 20 4 6O 80

EFFLUENT VOLUME (rnl)

Fig. I. Chromatography of enzymica l ly treated rabbit t h y m u s lysine-rich histone. Chromato- graphy was carried out on Amberl i te IRC-5o (i cm × 15 cm) with a l inear gradient (total volume, 14o ml) from 8. 5 to 14% guanidinium chloride in o.I M sodium phosphate , p H 6,8. Fract ions ot o. 7 ml were collected, and alternate fractions were analyzed for protein concentrat ion turbidi- metr ical ly 18. A. Untreated. B. Alkal ine phosphatase treated. C. Ribonuclease treated.

The phosphorylation of protamine has an effect on its chromatographic be- havior 4, and since lysine-rich histones are known ~ to be phosphorylated, chromato- graphic resolution and tissue specificity could be an expression of phosphorylation patterns. A dependence of histone phosphorylation on tissue source would be a simple extension of the notion s that phosphorylation plays a role in the derepression of genes. The latter notion has had significant support recently in the work of LANGAN, who demonstrated that histone phosphokinase may be stimulated by cyclic AMW and that histone phosphorylation in the liver is increased by insulin and glucagon 8.

Although earlier analyses a failed to detect RNA in chromatographically resolved lysine-rich histones, the binding of very low levels of RNA by histone with or without traces of nonhistone protein might have provided a basis for chromatographic reso- lution. Tissue specificity has been reported for chromosomal RNA 9. As reported below we have examined the action of ribonuclease on chromatographic patterns of lysine-

Biochim. Biophys. Acta, 221 (197 o) 128-131

130 SHORT COMMUNICATIONS

rich histones with the expectation that enzymic digestion would eliminate chromato- graphic resolution if it were based on RNA-complex formation. We have also tested the notion of RNA-complex formation by examining chromatographically resolved histones for uridine incorporation. To test for phosphorylation as tile basis for reso- lution, we chromatographed the histones after their incubation with alkaline phos- phatase.

Purified rabbit thymus lysine-rich histones were isolated according to the procedure of DE NooIj AND WESTENBRINK 1°. IO mg of purified histone dissolved in o. 4 ml of o.o 5 M Tris buffer, pH 7.6, was treated with o.I mg of either alkaline phos- phatase (E. coli, Worthington Biochemicals) or pancreatic ribonuclease (Boehringer- Mannheim) at 37 ° for 2 h. This is essentially the procedure used by ~IARUSHIGE el al. 4.

After incubation, 0.6 ml of 149/o guanidinium chloride in o.i M sodium phosphate buffer, pH 6.8, was added, and the mixture frozen until chromatographed. The results of this experiment are shown in Fig. I. I t is apparent that neither enzymic t reatment had any effect on the chromatographic fractionation. The enzymes were then assayed in the presence of rabbit thymus lysine-rich histone in order to see if the histone caused inactivation. Alkaline phosphatase was assayed in the presence or absence of lysine- rich histones (histone :enzyme = IOO :I) by the p-nitrophenyl phosphate method of GAREN AND LEVINTHAL 11. The enzyme had an activity of 40.2 and 34.4 units/rag in the presence or absence of histone, respectively. Ribonuelease was assayed according to KALNITSKY et al. 12, and showed virtually the same activity (175o units/rag) in the presence and absence of a Ioo-fold excess of lysine-rich histone. At an RNA to histone ratio of 50 :I the solution was turbid, but no precipitate settled out. At an RNA to histone ratio of IO:I the turbidity increased, but still no precipitate settled out. How- ever, at this increased ratio of histone to RNA, the apparent activity of the enzyme fell from 175o to 133o units/mg. This can be interpreted as complex formation between RNA and histone which was more slowly hydrolyzed by the enzyme during a standard incubation time of 4 rain.

Since complexes of RNA and histone might be more stable towards ribonuclease t reatment than free RNA, an a t tempt was made to label the lysine-rich histones of mouse mammary gland with nucleotide precursors in organ culture. The system of JUERGENS el al. 1~ was used. This is the same system that we have previously used to demonstrate that hormones may affect the incorporation of amino acids into specific subfractions of lysine-rich histone H. Explants of mammary glands from mice in mid- pregnancy were incubated in Medium 19q supplemented with 5 ~g/ml of insulin for 4 h in the presence of 25 #C/ml of ~3H]uridine. (Schwarz Biochemical Res., specific activity approx. 20 C/mmole). Other explants were incubated in a similar medium in the presence of 25 #C/ml [~H]thymidine (Schwarz Biochemical Res., specific activity > IO C/mmole) to serve as a control for nonspecific labeling of the histones. Lysine-rich histones were isolated from explants as previously described 17. No de- tectable radioactivity was observed in the histone from either incubation.

These studies support the conclusion that the fractionation of lysine-rich histones into species and tissue-specific patterns by ion-exchange chromatography is largely due to pr imary structural differences among the histone molecules rather than to phosphorylation of amino acid side chains or complex formation with RNA. These results are particularly interesting in the light of a recent report by SHEROD et al. ~5 that the electrophoretic heterogeneity of mouse ascites lysine-rich histone may

Biochim. Biophys. Acta, 221 (197 o) 128-i3z

SHORT COMMUNICATIONS 131

be reduced by treatment with alkaline phosphatase. Indeed, PANYIM AND CHALKLEY 16 have argued in favor of a microheterogeneity imposed upon a parent lysine-rich histone by differing levels of phosphorylation. Of course the two kinds of heterogeneity may coexist. The resolution by electrophoresis might be dominated by the difference in levels of phosphorylation while that of ion exchange could be dominated by differ- ences in amino acid composition and sequence. It is nevertheless important to recog- nize that most chromatographically resolved lysine-rich histones can also be dis- tinguished from each other electrophoretically 1,17 indicating that electrophoresis is sensitive to the differences in primary structure. Ongoing studies to determine the sequence of amino acids in certain subfractions of rabbit thymus lysine-rich histones should elucidate the structural differences.

This investigation was supported by grants from the U.S. Public Health Service (AM 12618, GM oo3I), the Agricultural Experimental Station, and Cancer Research Funds from the University of California, Berkeley. P.H. is the recipient of a fellowship from the Damon Runyon Memorial Fund for Cancer Research, Inc.

Department of Biochemistry, University of California, Berkeley, Cahf. 9472o (U.S.A.)

KATHERINE EVANS

PHILIP HOHMANN

R. DAVID COLE

I M. BUSTIN AND R. D. COLE, ,]. Biol, Chem., 243 (I968) 4500. 2 J. M. KINKADE, J. Biol. Chem., 244 (1969) 3375. 3 J. M. KINKADE AND R. D. COLE, J. Biol. Chem., 241 (1966) 5798. 4 K. MARUSHIGE, V. LING AND G. t{. DIXON, .[. Biol. Chem., 244 (I969) 5953. 5 M. G. ORD AND L. A. STOCKEN, Biochem. J., 98 (1966) 888. 6 L. J. KLEINSMITH, V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. dcad. Sci. U.S., 55 (1966)

1182. 7 T. A. LANGAN, Science, 162 (i968) 579. 8 T. A. LANGAN, Pro& Natl. 3cad. Sci. U.S., 64 (1969) 1276. 9 J. BONNER AND J. WIDHOLM, Proc. Natl. Acad. Sci. U.S., 57 (I967) 1379.

IO ];~. H. DE ~NOOlj AND H. G. K. WESTENBRINK, Biochim. Biophys. Acta, 62 (I962) 6o8. I I A. (;AREN AND C. LEVINTHAL, Biochim. Biophys. Acta, 38 (196o) 47 o. 12 G. KALNITSKY, J. I -). HUMMEL AND C. DIERKS, J. Biol. Chem., 234 (1959) I512. 13 \V. G. JUERGENS, ]3". E. STOCKDALP, Y. J. TOPPER AND J. J. ELIAS, Proc. Natl. Acad. Sci.

U.S., 54 (I965) 629. 14 P. HOHMANN AND R. 1). COLE, Nature, 223 (I969) lO64. 15 D. SHEROD, S. PANYIM, R. BALHORN AND R. CIIALKLEY, Federation Proc., 29 (I97 o) 73 o,

Abstr. I6 S. PANYIM AND R. CHALKLEY, Biochemistry, 8 (i969) 3972. 17 J. M. t{INKADE AND R. D. COLE, J . Biol. Chem., .241 (1966) 5790. i8 J. ~'~. LUCK, P. S. RASMUSSEN, i~. SATAKE AND A. N. TSETIKOV, .]. Biol. Chem., 233 (I958)

I4O3,

Received April 27th , i97o

Biochim. Biophys. Acta, 22i (i97 o) i 2 8 - I 3 I