- Email: [email protected]
Complexing of ATP with molybdate
voL. 1 8 ( I 9 5 5 )
SHORT COMMUNICATIONS, PRELIMINARY NOTES
6oi
Complexing of ATP with molybdate I n e x p e r i m e n t s on the effect of KCI concentration Oil the ATPase activity of m y o s i n we flmnd, as had been reported b y others, t h a t there was an o p t i m u m ATP concentration above which the ATPase activity decreased. \Vith increasing KC1 concentrations inhibition began at progressively lower ATP concentrations. F u r t h e r e x a m i n a t i o u of the s y s t e m showed t h a t this a p p a r e n t inhibition was due to the formation of a salt sensitive colorless complex between ATP ~nd molybdate. This complex did not become colored upon the addition of reducing agent and catalyst. Inorganic o r t h o p h o s p h a t e (Pi) was m e a s u r e d by the FISKE-SUBBAROW technique 1. Five ml of the sample to be analyzed were added to two ml of lO% trichloracetic acid and five ml of the m i x t u r e were t h e n added to one ml of 2. 5 % a m m o n i u m m o l y b d a t e in 3 N H2SO 4. Color d e v e l o p m e n t was initiated by addition of o. 5 ml of I-amino-2-naphthol-4-sulfonic acid (ANSA) in bisulfite solution 1. The optical densities were measured at 75o ni/, in a Beckman spectrop h o t o m e t e r ten m i n u t e s after the addition of ANSA. All c o n c e n t r a t i o n s of ATP, Pi, KC1, etc., refer to the original 5 ml test sample. I t was found t h a t Pi s t a n d a r d s had optical densities which were independent of the concent r a t i o n of KC1 (or NaC1). W'hen the o r t h o p h o s p h a t e blank izl v a r y i n g quantities of an ATP solution was m e a s u r e d it was found t h a t the a p p a r e n t blank decreased at high ATP concentrations. At o.o 5 3 I KC1 there is an a p p a r e n t loss of color at ATP concentrations greater t h a n 7' I°-3 M. Table I shows the m e a s u r e d Pi at two different concentrations of ATP as a function of KC1 concentration. I t is seen t h a t in the presence of high ATP concentrations the estimation of Pi can be in error even at low KC1 concentrations. In the presence of 8. IO-3 M ATP at o.o 3 M KC1, Pi standards gave color values a b o u t 35 % too low (contrasted to e x p e r i m e n t s w i t h o u t ATP), while in the presence of 4" t ° - a M ATP at o.6 M KC1 the color values were only a b o u t 5 % too low. TABLE I Increasing the a m o u n t of m o l y b d a t e in the OPTICAL DENSITY (O.D.) OF THE Pi IN T\¥O s y s t e m increased the optical densities near to SAMPLES OS ATP the expected values. We have obtained simil a r results with A D P and ITP. Although it KCl O.D. for Corrected*O.D. h a d previously been suspected2, 3 t h a t ATP moles/liter ro a M A T P [or5.ro -3 M A T P and o t h e r organic p h o s p h a t e s did complex with m o l y b d a t e , the present results show t h a t 2.o 5 o.488 at high KCI and at high ATP concentrations 1.o5 0.658 t.31 the complexing m a y be extensive enough to 0.55 o.768 [.3i affect the sensitivity of the FISKE-SUBBAROW 0.25 O,850 1.3 ° test unless proper precautions are observed. 0.05 0.938 1.3o I t seems possible t h a t some of the reported o (extrapolated) 0.96 1.32 salt dependent inhibitions of myosin ATPase b y high s u b s t r a t e concentrations m a y be artifacts of the analytical method. * Corrected O.D. = 2 × measured O.D. N a v a l M e d i c a l Research Inslitute, Bethesda, M d . ( U . S . A . )
JAcoB J. BLUM ROBERT W . CHAMBERS
1 C. H. FISKE AND Y. SUBBAROW, J . Biol. Chem., 66 (I925) 375. 2 H. \¥EIL-MALHERBE AND R. H. GREEN, B i o c h e m . J., 49 (1951) 286. 3 R. M. FLYNN, M. E . JONES AND F. LIPMANN, J. Biol. Chem., 211 (1954) 791Received October 8th, J955
Relations among cystine content, molecular weights and ultraviolet light sensitivity of proteins A relationship connecting q u a n t u m yields, O, (for inactivation of specifically active proteins) w i t h molecular weights, 7F/, of proteins has been found, n a m e l y 1 O = 0/31 = 447/M.
(i)
Recent work with other e n z y m e s 2 and antibodies s has s u p p o r t e d the equation. SETLOW4 has suggested a n o t h e r relationship*, n a m e l y t h a t q u a n t u m yields are r o u g h l y p r o p o r t i o n a l to the cystine c o n t e n t of enzymes, If b o t h propositions are correct, one would expect to find t h a t molecular weights of proteins and reciprocals of percent a b u n d a n c e of cystine tend to be directly related.
SHORT COMMUNICATIONS, PRELIMINARY NOTES
6oi
Complexing of ATP with molybdate I n e x p e r i m e n t s on the effect of KCI concentration Oil the ATPase activity of m y o s i n we flmnd, as had been reported b y others, t h a t there was an o p t i m u m ATP concentration above which the ATPase activity decreased. \Vith increasing KC1 concentrations inhibition began at progressively lower ATP concentrations. F u r t h e r e x a m i n a t i o u of the s y s t e m showed t h a t this a p p a r e n t inhibition was due to the formation of a salt sensitive colorless complex between ATP ~nd molybdate. This complex did not become colored upon the addition of reducing agent and catalyst. Inorganic o r t h o p h o s p h a t e (Pi) was m e a s u r e d by the FISKE-SUBBAROW technique 1. Five ml of the sample to be analyzed were added to two ml of lO% trichloracetic acid and five ml of the m i x t u r e were t h e n added to one ml of 2. 5 % a m m o n i u m m o l y b d a t e in 3 N H2SO 4. Color d e v e l o p m e n t was initiated by addition of o. 5 ml of I-amino-2-naphthol-4-sulfonic acid (ANSA) in bisulfite solution 1. The optical densities were measured at 75o ni/, in a Beckman spectrop h o t o m e t e r ten m i n u t e s after the addition of ANSA. All c o n c e n t r a t i o n s of ATP, Pi, KC1, etc., refer to the original 5 ml test sample. I t was found t h a t Pi s t a n d a r d s had optical densities which were independent of the concent r a t i o n of KC1 (or NaC1). W'hen the o r t h o p h o s p h a t e blank izl v a r y i n g quantities of an ATP solution was m e a s u r e d it was found t h a t the a p p a r e n t blank decreased at high ATP concentrations. At o.o 5 3 I KC1 there is an a p p a r e n t loss of color at ATP concentrations greater t h a n 7' I°-3 M. Table I shows the m e a s u r e d Pi at two different concentrations of ATP as a function of KC1 concentration. I t is seen t h a t in the presence of high ATP concentrations the estimation of Pi can be in error even at low KC1 concentrations. In the presence of 8. IO-3 M ATP at o.o 3 M KC1, Pi standards gave color values a b o u t 35 % too low (contrasted to e x p e r i m e n t s w i t h o u t ATP), while in the presence of 4" t ° - a M ATP at o.6 M KC1 the color values were only a b o u t 5 % too low. TABLE I Increasing the a m o u n t of m o l y b d a t e in the OPTICAL DENSITY (O.D.) OF THE Pi IN T\¥O s y s t e m increased the optical densities near to SAMPLES OS ATP the expected values. We have obtained simil a r results with A D P and ITP. Although it KCl O.D. for Corrected*O.D. h a d previously been suspected2, 3 t h a t ATP moles/liter ro a M A T P [or5.ro -3 M A T P and o t h e r organic p h o s p h a t e s did complex with m o l y b d a t e , the present results show t h a t 2.o 5 o.488 at high KCI and at high ATP concentrations 1.o5 0.658 t.31 the complexing m a y be extensive enough to 0.55 o.768 [.3i affect the sensitivity of the FISKE-SUBBAROW 0.25 O,850 1.3 ° test unless proper precautions are observed. 0.05 0.938 1.3o I t seems possible t h a t some of the reported o (extrapolated) 0.96 1.32 salt dependent inhibitions of myosin ATPase b y high s u b s t r a t e concentrations m a y be artifacts of the analytical method. * Corrected O.D. = 2 × measured O.D. N a v a l M e d i c a l Research Inslitute, Bethesda, M d . ( U . S . A . )
JAcoB J. BLUM ROBERT W . CHAMBERS
1 C. H. FISKE AND Y. SUBBAROW, J . Biol. Chem., 66 (I925) 375. 2 H. \¥EIL-MALHERBE AND R. H. GREEN, B i o c h e m . J., 49 (1951) 286. 3 R. M. FLYNN, M. E . JONES AND F. LIPMANN, J. Biol. Chem., 211 (1954) 791Received October 8th, J955
Relations among cystine content, molecular weights and ultraviolet light sensitivity of proteins A relationship connecting q u a n t u m yields, O, (for inactivation of specifically active proteins) w i t h molecular weights, 7F/, of proteins has been found, n a m e l y 1 O = 0/31 = 447/M.
(i)
Recent work with other e n z y m e s 2 and antibodies s has s u p p o r t e d the equation. SETLOW4 has suggested a n o t h e r relationship*, n a m e l y t h a t q u a n t u m yields are r o u g h l y p r o p o r t i o n a l to the cystine c o n t e n t of enzymes, If b o t h propositions are correct, one would expect to find t h a t molecular weights of proteins and reciprocals of percent a b u n d a n c e of cystine tend to be directly related.