Mechanism of methyl phosphate formation by “killing” of spinach chloroplast fragments with methanol in the presence of phosphate

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EIOCHIMICA ET BIOPIiYSICA ACTA

Preliminary Notes Me.c~nism of metl~l pEm,Sl~ate formation by "killing" of spinach c~loro~s~ fr'~gmenl:swith methanol in the presence of phosph~,e A previous p a p ~ ~ trom our laboratory dealt with the identification of monometbvl ph,.~:pha~e me a produc~ fformed when spinach chloroplast fragments, incubated with "~P~:, were t r e a t e d ~ S t h e x c e s s m e t h a n o l , in o r d e r t o s t o p p h o ~ o p h u s p h o r y l a t i o n a n d o~daer enm-n.-Ac ]proc~s_~es. T h i s fformation of me*thx-] p h o s p h a t e w a s i n t e r p r e t e d as dotro s o m e " a c t i v e " p h o s p h o ~ - l a x i n g agenx, b u i l t u p during~ t h e i n c u b a t i o n , whi,-h c o u l d rr.ansier J~s ph:~--i~hai*:group zo The added alcoho] in a non-enzymic rea,:tio~, ~. In ,~h~,course of conninued mve~imafion, however, we found no evidence f(,¢the cxi~ten,.TM, of T.he posTulaze,a pho.wphaze d o n o r , b m zha~ there: i-~ s u m ~ p h u s p t m z a , ~ c a n ; a ~ e d i~ -,-he c ~ o r o p l a . ~ s , w h i c h c a z A v s e s a r e s t e r i f i c a t i o n e q t f i h b r i u m betweev, pho.%phaTe a n d m e t . k , ~ o ] on t h e o n e h a n d e n d mexhxfl pho~-ph~te a n d w a t e r o n tiv: ozhe:'. Thi.~ p h o s p h a z a s e o f t e n r e m a i n s efleuxivv in u p 10 ~o Of al~x)ho! or ~enai.n uti~er .qr~,~l~ic s o ) v t ~ 1 s al r o o m lemY~a'~uyt.

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PRELIMINAR*/' NOTES

191

The equilibrium could be approached from both sides, starting with phosphate as initial compound as well as with methyl phosphate (Fig. I). Very pure chloroplasts, isolated from frozen-dried spinach leaves by a nonaqueous procedure (combination of methods described in refs. 2 and 3), gave similar results (in agreement with U. HEBER, private communication), indicating that the reaction is unlikely to be due to an impurity contained in the chloroplast preparation. The reaction is also given by commercial acid and alkali pllosphatases (in agreement with E. TYSZKIEWICZ,private communication), Chlorella cells and rat blood. This indicates that there are a number of phosphatases in biological materials which have a similar high stability. The earlier observation1 of the appearance of some methyl phosphate upon the treatment with methanol of a mixture which had been heated after incubation is an error, probably due to incomplete heating so that a small part of the enzyme survived. The preparation of this paper was sponsored bv the U.S. Atomic Energy Commission. Del~.arlment of Chendstrv and Lawrence Radiation Laboratory, University of California, Berkeley, Calif. (U.S.A.)

JOHANNESULLRICH* ~IELVIN CALVIN

t E. TYSZKIEWICZ,G. GINGRASANn M. CALVIN,Biochim. Biophys..4eta, 50 (I961) 376. 2 U. HEBER, Ber. deut. botan. Ges., 7° (rtJ57) 37[. 3 C. R. STOCKING, Plant Physiol., 34 (I959) 56. Received December 8th, 1961 * NATO fellow I061-62, on leave from Universi~: of Bonn (Germany), Department of Chemistry. Biochim. Biophys. Acta, 57 (I962) 19o-I9r

Porphyrin biosynthesis Studies on the nature of a biosynthetic porphyrin and its identification with the so-called "208" porphyrin "Porphyrin 2o8" is ~ hepta-carboxylic, isomeric type-III porphyrin, which has been identified in porphyria material. It was isolated and described as a chemical entitl~ forming a significant component of a porghyrin complex1, the uroporphyrin III of WALDm~STROM2 later named type A, WALD~NSII~6M porphyrin3. It is present in significant quantities in the urine of patients with porphyria hepatica, particularly of the cutanea tarda type a. FALK et al. 5-r have isolated, after incubation of chicken red-cell preparations with porphyrin precursors, a porphyrin which they have named "pseudouroporphyrin". On the "dioxane" paper chromatograms of FALK a,nD BENSONs this porphyrin runs as a. discrete spot, faster than uroporphyrin III, and on decarboxylation it yields coproporphyrin I I I only5-7. A porphyrin of m.p. 2IZ-216 ° isolated by CANIVET AND RIMINGTON9 from c~taneous porphyria urines behaves identically on these chromatograms ~,9. A sample of the original "208" porphyrin which was available Biochim. Biophys. ,4cta, 57 (r962) r9x-z94