Oct. 5 , 195.5
5105
COMMUNICATIONS TO TIIE EDITOR
TABLE I1 REACTIONS CATALYZED BY THIOLASE 110-fold purified heart thiolase (specific activity 37 when prepared). Final concentrations: 0.01 J4 TRIS-HC1 buffer PH 8.1, 1.0 x 10-4 M ACAC-SR, 2.0 x 10-4 M R-SH, M MgC12, 1.5 X 10-3 M Ac-S-pantetheine. 5 X Specific activity
Reaction
AcAc-S-COA $- C O A S H 4.05 AcAc-S-Pantetheine CoASH 3.02 AcAc-S-Pantetheine pantetheine 0.80 AcAc-S-CoA pantetheine .i 9 2-Ac-S-pantetheine , 0gc Ac-S-Pantetheine" CoA-SH .15 AcAc-S-AC-N-PMEA~ HS-AcK-/3MEAb 0 1.0 X 10 - 3 M ; pH 7 . 5 ; spontaneous rate was 0.2 times Indirect assay" the enzymatic one. b 3.3 X 10-3 114. pH 7.1. By direct assay ( p H 9.2) 0.035.
+
+ +
+
+
I n liver extracts pantetheine did not substitute for CoA in the aceto-CoA-kinase reaction13 nor in the butyro-CoA-kinase r e a ~ t i 0 n . l ~However, the synthesis of A~-S-pantetheine'~ from Ac, -4TP and pantetheine occurs in pigeon liver extract provided catalytic amounts of CoA-SH are added. This indicated the coupling of the aceto-CoAkinase (reaction 7) with reaction 8 (the reverse of reaction 5 ) :
CoA fatty acid cycle. The demonstration of enzymes which synthesize pantetheine thioesters is further evidence for the occurrence of a pantetheine fatty acid cycle. Unlike the Coil cycle, the pantetheine cycle is not directly linked to the citric acid cycle; and, since crotonase appears to be the ratelimiting step in this cycle, i t may play a role in isoprenoid synthesis. The relative amount of CoA and pantetheine in bacteria and tissues, l 9 and hence the quantitative significance of the respective cycles, has still to be assessed. (19) E E Snell and G M Brown, Advances t n E Z Z Y ~ O ~ O 14,E 40 Y,
(1933)
DEPARTMENT OF PHARMACOLOGY
SCHOOLOF MEDICINE
JOSEPH
R STERN
WESTERN RESERVE UNIVERSITY 6, OHIO CLEVELAND RECEIVED JULY 12, 1955 SIMILARITIES BETWEEN SO-CALLED CHLOROPHYLL b" AND OXIDIZED CHLOROPHYLL b, AND BETWEEN SO-CALLED CHLOROPHYLL a" AND OXIDIZED CHLOROPHYLL a
Sir:
Heat, alkalies and oxygen convert chlorophylls a and b into several chromatographically unique pigments that are spectrally similar to the parent chlorophylls. Some of these pigments, chlorophylls Ac CoA-SH 4-ATP a' and b', are isomers or other reconvertible alteraAc-S-COA E-AMP PP ( 7 ) tion products of the common chlorophylls.' Some Ac-S-CoA pantetheine-SH are oxidation or allomerization products that have Ac-S-pantetheine -I- CoA-SH (8) not been reconverted to the chlorophylls.2 Others, The reversibility of reaction 8 was shown by chlorophylls a" and b", are regarded as chlorophyll coupling i t with the crystalline citrate condensing isomers although they also resemble the nonenzyme and purified malic dehydrogenase16 and reconvertible allomerized chlorophyll^.^ Addiobserving a CoA-SH dependent reduction of DPN tional indications that a" and b" are oxidation in the presence of Ac-S-pantetheine, L-malate, and products are presented herein. pigeon liver extract. The specific activity was I n repetition of the experiments of Strain and 0.014. The enzyme catalyzing reaction 8 belongs Manning' and of Strain,2 heat or traces of alkali to the class of thioltransacetylases recently de- converted chlorophyll a, in n-propyl alcohol soluscribed in pigeon liver by Brady and Stadtmanl? tion, into a' plus a. The less sorbed a', isolated who did not test pantetheine. Pigeon liver ex- in a column of powdered sugar and redissolved in tracts were also found to catalyze a CoA-SH de- propyl alcohol, was reconverted into a plus a' by pendent hydration of CROT-S-pantetheine to d- heat or alkalies. These preparations of a and a' BOK-S-CoA (specific activity 0.0056) indicating were readily interconverted again by heat or alkathe presence of a thioltranscrotonylase catalyzing lies. Consequently, a and a' are isomers or other reaction 9 readily interconvertible products. In similar experiments, chlorophyll b yielded the reconvertible, CROT-S-pantetheine CoA-SH less sorbed b'. Neither a nor b yielded more CROT-S-CoA pantetheine-SH (9) sorbed green pigments. Since liver also catalyzes a stepwise conversion Freed, et a1.,3 found the strongly sorbed b" of sorbyl-S-CoA through P-ketohexenoyl-S-CoA (yield ca. 14%), in addition to weakly sorbed b', to CROT-S-CoA and AC-S-COA,'~ the exact rela- when very dilute propyl alcohol solutions of b were tion of the thioltransacylases present in liver ex- heated in vacuum. This pigment was regarded as tract to thiolase-type enzymes remains to be de- a chlorophyll, because i t was prepared in vacuum, t ermined. its spectral properties resembled those of b, and it It would seem that, as with crystalline crotonase, was more sorbed than b. Freed's allomerized b the pantetheine thioesters serve as substrates a t (method of preparation not described) was less physiological concentrations for the enzymes of the sorbed than b. Unlike b and b', b" did not give (13) M .E. Jones and F. Lipmann, in S. P. Colowick and N. 0. the phase test, and it was not reconvertible to b Kaplan, Methods in Enzymology, New Yovk, 1, 585 (1955). plus b'. (14) H.R. Mahler, S. J. Wakil and R. M. Bock, J . Bioi. Chem., 404, Strain2 has found, however, that b yields a par453 (1953).
+
+
+
+
+
+
(15) Optical assay (X240) according t o E. R. Stadtman. ibid., 4OS, 501 (1953). (16) J. R. Stern, S. Ochos and F. Lynen, ibid.. 198, 313 (1952). (17) R. 0. Brady and E. R. Stadtman, ibid., 411, 621 (1954). (18) J. R.Stern, unpublished observations.
(1) H. H. Strain and W. M. Manning, J. Bioi. Chem., 146, 275 (1942). ( 2 ) H.H.S t a i n , Aguicullural and Food Chem., 2, 1222 (1954). (3) S. Freed, K. M. Sancier and A. H. Sporer, THISJOURNAL, 76, 6006 (1954).
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COM;\IUNICATIONS TO THE EDITOR
ticular oxidation product that is similar to b”. Allomerized in methanol or in barley leaves, b yields several pigments all more sorbed than b. One of these is spectrally similar to b (A max. methanol, b, 652 mp; oxidized b, 633 mp). This product, therefore, resembles b ” both spectrally and chromatographically. Like b” it is not isonierized by heat or alkalies; and it does not give the phase test. Except for the method of preparation, this allomerized b is identical with b”. The a”, for which few properties have been determined,3 has its counterpart among the oxidation products? of a. The increased sorbability of the oxidized chlorophylls2 and of a “ and b ” relative to a and b may be attributed to additional oxygen (or decrease in hydrogen) . 4 Molecular structures proposed by Freed, et a1.,3 for the chlorophylls and their isomers are unsuitable. If a, a‘, and oxidized a have the magnesium atom bound to different pairs of pyrrole nitrogen atoms,3 the three pigments should yield but one magnesium-free pheophytin. Yet, three have been reported.’!? Moreover, oxidized chlorophylls, presumably with magnesium bound as in the chlorophylls, do not undergo isomerization.2 The strongly sorbed pigments prepared by Freed, et al., and by Strain are oxidized chlorophylls, not isomeric chlorophylls. These products should not be called chlorophylls (unless found as natural constituents of plants).
Vol. 77
TABLE I THREETIMESRECRYSTALLIZED ~I.U?.A~IIC DEHYDROGENASE OF BEEF LIVER
~hSAI.I.SES O F
Element
Preparation 1 GDW
(pg./g.
Prep:rration 3 GDW
(pg./g.
Ziiic 333 ntl. I, Copper (r Aluminum (73.1 Barium 27.0 on. n b Calcium 1060 Magnesitim 149 43.0 * Xot determined. b Not detected, and also beryllium, cadmium, cobalt, chromium, iron, lithium, molybdenum, manganese, nickel, potassium, silver, strontium, tin and lead.
Fractionation of beef liver shows an aggregation of zinc with purification of the enzyme, and as the activity : protein ratio reaches its maximum value. The meta1:protein ratio of all other elements studied decreases with progressive enzyme purification. The activity: zinc ratio increases progressively with purification, reaching a high value in the third crystals. The rate of conversion of DPN to DPNH a t #H 1 . 1 in the presence of glutamate’ is inhibited significantly when the enzyme is preincubated with a number of metal binding agents, including sodium sulfide, sodium diethyldithiocarbamate and 1 , l O phenanthroline. These data establish glutamic dehydrogenase as a zinc metalloenzyme according to the criteria published elsewhere. ( 4 ) H. H. Strain, ”Chromatographic Adsorption Analysis,” Interscience Publishers, Inc., New York, S . lr.,1942, pp. 14-22, The oxidation of glutamic acid is the second DPN dependent dehydrogenation reaction known CHEMISTRY DIVISION A R G O N N E NATIONAL LABORATORY HAROLD FI. STRAIN to be catalyzed by a zinc metalloenzyme.? 1,EXONT. IL1,INOIS Acknowledgments.-The valuable suggestions of RECEIVED JULY 22, 1955 Dr. Eric G. Ball are gratefully acknowledged. This work was supported by grants-in-aid from the GLUTAMIC DEHYDROGENASE OF BEEF LIVER, A Office of Naval Research, Contract No. NR 119277, to Harvard University, and by the Rockefeller ZINC METALLOENZYME Foundation, New York, N. Y. Sir: LABORATORY BERTL. VALLEE LX’e recently have examined several crystalline BIOPHYSICSRESEARCH THE DEPARTMENT OF MEDICINE S. JAXES A D E L S T E I ~ preparations of beef liver glutamic dehydrogenase OF HARVARD MEDICALSCHOOL ASD PETER and find that zinc is an integral and functional BEXTBRIGHAM HOSPITAL, BOSTON,MASS. component of the enzyme molecule. JAMES A . Or,sos DEPARTMENT OF CHEMISTRY The protein was crystallized from fresh beef liver.’ HARVARD UNIVERSITY MASS. Quantitative emission spectrography and micro- CAMBRIDGE, chemical determination of zinc were performed in ( 3 ) B. I,. Vallee, “Zinc and Metalloenzymes” in “Advances i n Produplicate according to methods previously de- tein Chemistry,” Vol. X, Academic Press, New York, K. Y . , 19.L?,i l l scribed.2 Protein weights were determined by press. (4) Fellow of t h e Xational Foundation for Infantile Paralysis. trichloroacetic acid precipitation. Table I presents quantitative spectrochemical and microchemical data on three times crystallized STEROIDAL SAPOGENINS. XXXI. GENTROGENIN preparations of glutamic dehydrogenase having AND CORRELLOGENIN, NEW SAPOGENINS FROM Dioscorea spicutiforal high specific activity. Preparation 1 contained 333 p g , Zn/g. of protein, preparation 2 contained S i r : 322 pg. Zn/g. of protein, and a third preparation Some years ago Marker announced the isolation contained 260 pg./g. The zinc content of these of botogenin and neobotogenin from D. mexicrystalline preparations was not lowered by pro- ~ a m . ~The a ~ structures ~ assigned had both a 12longed dialysis. The molecular weight of glutamic keto and a 5,G double bond. Since substances with dehydrogenase has been determined to be about these groupings would be particularly desirable as one million.’ Based on this molecular weight, the cortisone precursors, there was an extensive but number of gram atoms of zinc per mole of enzyme fruitless search made for them. mTe have now protein vary between 4 and 5 for these preparations. found in several collections of D. spiculiflora (1) J. A. Olson and C. B. Anfinsen, J . Bid. Chem., 197,67 (1952). (2) B. L. Vallee and F. L. Hoch, Proc. Nail. Acad. Sci., 41, 327 (195,j).
3 -
(1) Paper XXX: XI. E Wall, submitted in E i p e r i e n f i a . ( 2 ) (a) R. E. Slarker and J. Lopez, THISJOURNAL, 69,2397 (1947); (b) R. E. Marker, ibid., 71,2650 (19.19).
