COMMUNICATIONS TO THE Eurruw
Feb. 20, 1960
63
TABLE I INDKCTION OF CRYSTAL FORMATION BY COLLAGEN Determination of the point of precipitathn was accomplished by techniques published earl$r (Fig. 1, ref. 5 ) . Conditions were p = 0.16; t = 3'7 ; PH 7.4; tinie of equilibration, 3 days. ---Point so. expts.
Mean
50
of precipitationStandard Standard deviation deviation of mean
Control solutions 7 50 4.1 Collagen-seeded 11 20 4.7 a Expressed as Ca X P, in (mg. % j z .
1.6 1.4
4';
(397
-
j
r- i
4
t-
REGIOS OF mTASTA6I LITY
1
I
L-
I
CB x
pi
PRECIPITAYIUN
'
KSp
CaHPO+
REGION OF LTDERSAT'Y I N ABSENCE OF
i
CATALYZED INDUCTION
SERUM LEVELS Presumably, this phenomenon results from localSOLUBILIZING ACTION ized acid production by the living bone cells themOF CELL SECRETION selve~.~.~ In the absence of preformed solid phase, it is the I Ksp of CaHP04.2H20 which seems to govern the B O W hlINERAL stability of the aqueous calcium: phosphate ~ y s t e r n . ~ Spontaneous precipitation never has been described sutmnary of prcserit inforn~ationcmiccrning Fig l.-!i in solutions having products, Ca X P i , less than diisolution and formxtion o f hone niineral given in terms of about 35 (mg. % ) 5 although varying degrees of metastability have been observed (depending the serum prcduct, Ca X P,, in (mg. yo)*. upon conditions) with solutions having products of physiological concentrations of calcium and as high as 50. These general findings are sum- phosphate. These data are summarized in Table marized in Fig. 1. 1. Until now the idea that collagen can function as The case for collagen now seems complete. It a crystal nucleator in vivo has suffered from a serious can specifically cause crystal formation under quantitative defect. Crystal induction by col- physiological conditions. Whether it does this in lagen has been demonstrated in several labora- viao and how it accomplishes this remain questions has been shown to be yet to be answered by further research. t o r i e ~ . ~ s ~The ~ , ~process l exquisitely specific4J0 and morphologically the DEPARTMENT OF RADIATION BIOLOGY OF MEDICINE A N D DENTISTRY early phases of crystal induction are very similar SCHOOL HERBERT FLEISCH OF ROCHESTER both in vivo and in v i t r ~ . ~Nonetheless, ~~'~ until THEUNIVERSITY ~VILLIAMF. XEUMAN 20, SEW YORK the present, these examples of crystal induction ROCHESTER RECEIVED J A N U A R11, Y 1960 have all involved solutions in the region of metastability (>K,, CaHP04.2ELO) involving products of Ca X Pi from 35 to 50 (mg.%)2.3t5J1J3Yet to PROTON RESONANCE SPECTRUM AND STRUCTURE be functional in the living animal, collagen must OF THE AZULINEUM I O N be shown to induce crystal formation a t physiologi- Sir: cal products (ca. 20), a region of undersaturation The structure of the azulinium ion is of considerwith respect to solid formation, able interest in concection with the electron charge Quite recently,I4 crystal induction has been distribution in azulene itself. Protonation of demonstrated with demineralized, fresh dentin azulene in acid solutions may be expected to occur a t products, Ca X P i , of 20, well below the critical a t the 1 and 3 positions, which, according to K,, CaHP04.2H20. This represented the first molecular orbital calculations,l have the greatest occasion, to our knowledge, of crystal nucleation excess a-electron charge density. It is also known by a non-living system under physiologically mean- that the protons a t the 1 and 3 positions of azulene ingful conditions. The tissue residue employed, are readily exchanged in D,S04 solutions.2 We however, undoubtedly contained substances other have now been able to confirm the structure of the than collagen. Hence, the case for collage11 as azulinium ion on the basis of a high-resolution the nucleator, though greatly strengthened in a proton resonance spectrum. A previous attempt3 quantitative sense, was still not proven. In experi- to determine the structure from the proton resoments just completed, a collagen'j prepared by the nance in concentrated sulfuric acid was unsuccessful method of Einbinder and SchubertlG also has due to inadequate resolution. proved able to cause crystal formation in solutions Figure l a shows the proton resonance spectrum (9) G. R. Martin, H. E. lirschein and W.I'. Keumun, THIS JOUR- of a 7 mole % solution of pure azulene in CCL. N A L , 80, 6201 (1958). A scale in c.!s., referred to the resonance signal of (10) M. I. Glirncher, A. 1. Hodge and IF. 0. Schrnitt, Proc. K a f l . CH2C12 (dotted line), is shown a t the bottom of the A c a d . Sci. U .S., 43, 860 (1957). figure. The assignment and analysis of the azu(11) B. N . Uachra, A. E. Sobel and J. W.StanFord, A r c h . Biochem. B i o p h y s . , 84, 79 (1959). lene proton spectrum has been described pre(12) S. Vitton Jackson and J. T. Randall, " R o n c Structure and viously.4 Shown 011 the sanic' scale ill Fig. l b is hIetaboli5ni," Ciba Fotinii;ition Symposium, ed. G . E. IV. W ~ ~ l ~ t c n l i o l i n e the proton resoiiance spectruiii of a '7 iiiole yo and C. U. O'Connor, London, 1956.
I
( 1 3 ) hl. Lanini and W. 17. Neuman. A r c h . Z ' u f h d , 66, 20.1 (!!l38), (11) C . C. Svlonions and W. F. Neutiiiiri, in preparatic,n. (1:) Upon analysis, this prcyaration was e5bentially C;r free, certainly less than 0.1 p . p , m .
(16)
I. Einfinder and
M. Schubert,
J. B i d . Chem., 188, 335 (1951).
(1) R . Pat iser, J . ' ~ ~ P I I PZ h. y s . , 25. I I l ? (ill3i). ( 2 ) J3. IIcilbrotiner, private cotntii~itiicati~~n. ( 3 ) H. hI. Prey, J . C h e m Phys., 25, GUI) (196fj). (4) W. G. Schneider, H. J. Eerristein and J. A . Poplc, NAL, 80, 3497 (1958).
1'111sJ U I , K -
998
Vol. 82
COMMUNICATIONS TO THE EDITOR
(a)
Fig. 1.-Proton
magnetic resonance spectrum a t 80 hlc./s. of ( a ) 7 mole 5; solution of azulene in CCla, and ( b ) i mole solution of azulene in CF3COOH. External reference signal (dotted line) is CH2C12.
solution of azulene in trifluoroacetic acid. It is evident that under these conditions the azulene is converted completely to the protonated ion. The signal a t high field, +93 c../s., is characteristic of methylene protons which are assigned to the 1 (or 3) position. The quadruplet centered a t -117 c./s., which is equal in intensity to that of the methylene proton signal, is a typical AB spectrum for two non-equivalent protons and arises from protons 2 and 3 on the 5-membered ring. 4 partially resolved spin-coupling (J-1 c./s.) between these protons and the two methylene protons is evident. The chemical shifts of protons 2 and 3 are only slightly altered from their values in pure azulene. The corresponding changes in the signals of the protons in the 7-membered ring are more pronounced; in the ion these protons tend to be more nearly equivalent and show a marked decrease in screening. These changes are consistent with the conclusion that the electron deficiency in the ion resides almost exclusively on the 7-melnbered ring, where it tends to be shared with all the carbon atoms in the ring. In the formula in Fig. lb, as suggested by Heilbronner and Sinionetta,: a positive charge is indicated as localized on the “branch” carbon atom, but may in fact be more delocalized in the 7-membered ring and tend to approach the configuration of the tropylium ion. From the width of the proton resonance lines it can be concluded that the mean “life-time” of the protonated azulene ion is a t least of the order of one second and probably greater. DIVISIOSO F P U R E CHEMISTRY S A T I O N A L IZESEARCH C O U S C I I ,
(A,
COMPETITIVE INHIBITION O F ENZYMATIC REACTIONS BY OXAMYCIN
Sir:
s.
Inhibition of a w e u s by oxaniycin ((D)cycloserine, (D) 4-amino-3-isoxazolidone), which results in accumulation of the uridine nucleotide, UDPGNXc-lactyl-(L) ala- (D) glu- (L)lys, is competitively reversed by (D)alanine.* The antibiotic is a struc(D)tural analog of this natural substrate. alanine is formed and incorporated into a uridine nucleotide through the reactions3 pyridoxal phosphate Eq. 1 (L)ala Eq. 2
--+
(D)ala
BTP
( d a h -+ ( ~ ) a l a - ( ~ ) a l a l Z h +-
(L)lys-(D)ala-(D)ala,
‘The purpose of this communication is to report that oxamycin is a competitive inhibitor of the enzymes catalyzing reactions 1 and 2. Reaction 1 is catalyzed by alanine r a ~ e m a s e . ~ Oxamycin is a competitive inhibitor of the reaction in both directions (Fig. 1). The Michaelis constants (K,) for (D)alanine ( G . l X 10-31W) and for (L)alanine (6.5 X 10-3-11) are nearly identical. Similarly, Ki for oxamyciri measured in the direction of (D)alanine formation (0.6 X 10-4J1) is
S. S. DANYLUK (1) Abbre>iations: C D P , uridine diphosphate; GSAc-lactyl, an W. G. SCHNEIDER ether of acetylglucosamine and lactic acid (acetylmuramic acid). (2) J. I,. Strominger, K . H. Threnn and S . S. Scott, T ~ n JsO T : R N A L , RECEIVED I~ECEMZR 2 2E, R1959
orTA\t-A, CASADA
( R ) E. Heilbronner and n i . Sirnonetta, Hcls. Chim. Acta, 35. 1049 (1952).
81,3803 (1959). (3) E. Ito and J. L. Strominger, J . Bioi. Chent., 236, Feh., (19ciO). (4) 11” A . Wood and I . C. Gunsalus, ibzd., 190, 403 (1951).
