- Email: [email protected]
Nicotinamide-adenine dinucleotide dehydrogenase activity of human erythrocyte membranes
165
SHORT COMMUNICATIONS
kidney 12, rabbit brain 13, at low concentration, and at higher concentrations in individual experiments in embryo chicken heart 14. Several explanations for these phenomena exist. We have earlier put forward a hypothesis based upon supposition of a conformational effect of ouabain upon the enzyme itself relative to a metal-binding site z°. This would suppose that ouabain affects the enzyme conformation in a manner which the pre-reaction conformation of the protein disposes. In consequence, availability of the binding site for potassium might be made greater or less by a single action of ouabain. This thesis may be economically applied to the present observations. This work was supported in part by U.S. Public Health Service Research Grant H E 09 571; in part by a Grant-in-Aid from the American Heart Association.
Biochemistry Department, University of Texas Medical Branch, Galveston, Texas (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13 14
HARRY DARROW BROWN
A. SCHWARTZ, Biochem. Biophys. Res. Commun., 9 (1962) 3Ol. J. C. SKOU, Biochim. Biophys. Acta, 23 (1957) 394J. C. SKOU, Physiol. Rev., 45 (1965) 596. T. Z. CS~KY, Rev. Physiol,, 27 (1965) 415 . K. AH~aED AND J. D. JUDAH, Canad. J. Biochem., 43 (1965) 877. M. V. RILEY, Exptl. Eye Res., 3 (1964) 76. A. SCHWARTZ AND A. H. LASETER, Biochem. Pharmacol., 13 (1964) 337. A. ASKARI AND J. C. FRATANTONI, Biochim. Biophys. Acta, 71 (1963) 229. L. E. HOKIN AND D. REASA,. Biochim. Biophys. Acta, 9o (1964) 176. H. D. BROWN, ~h~. J. 2~-EUCERE, A. M. ALTSCHUL AND W. J. EVANS, Life Sci., (1965) 1439. K. S. LEE AND D. H. YU, Biochem. Pharmacol., 12 (1963) 1253. R. F. PALMER AND B. R. NECHAY,J. Pharmacol. Exptl. Therap., 146 (1964) 92. S. L. BUNTING, N. M. HAWKINS AND M. R. CANADY,Biochem. Pharmacol., 13 (1964) 13. R. L. KLEIN, Biochim. Biophys. Acta, 73 (1963) 488.
Received September 27th, 1965 Revised manuscript received November i2th, 1965 Biochim. Biophys. Acta, 12o (1966) 162-165
BBA 43 077
Nicotinamide-adenine dinucleotide dehydrogenase activity of human erythrocyte membranes Evidence accumulated in the last few years supports the concept that the cell membrane is a metabolically active structure, where a "vectorial" assembly of enzymes may be closely related with transport processes 1. A number of enzymic activities has been found in membranes of bacteria and liver cells2,3. In red blood cell "ghosts", (Na+-K+-MgZ+)-activated ATP phosphohydrolase (EC 3.6.1.3) (ref. 4), AMP aminohydrolase (EC 3.5.4.6) (ref. 5), D-glyceraldehyde-3-ph°sphate:NAD oxidoreducta~e (phosphorylating)-ATP:D-glyceraldehyde 3-phosphotransferase complex (EC 1.2.1.12 and 2.7.I.28), together with a triple enzyme sequence of pentose phosphate pathway*, have been described. Also, incorporation of fatty acids into phospholipids has been detected in erythrocyte ghosts 7. Other findings seem to indicate that the red-cell membrane has, as a feature in common with other biological membranes, Biochim. Biophys. Acta, 12o (1966) 165-169
166
SHORT COMMUNICATIONS
a structural 8 and a contractile protein 9. Electron transfer reactions have been shown to proceed only in mitochondria and microsomal membrane. We were interested in investigating the mammalian cell membrane for the occurrence of enzymes of electron transfer reactions that presumably could play a role as energy devices in the plasmalemma. In this work we report evidence that red-cell membranes are able to oxidize NADH using different electron acceptors. Red blood cell ghosts were prepared from fresh venous blood of normal human adults according to DANON, NEVO AND MARIKOVSKI1° o r POST e¢ al. 4. The membranes obtained by DAXON'S procedure had a white color (99-95 % of the hemoglobin had been removed) and they appeared to be intact under the phase-contrast microscope; when the technique of POST was used they were shown to be fragmented. Red blood cell ghosts were kept in o.I M histidine-imidazole buffer (pH 7.1) at 4 ° for a period not longer than 4 days. Protein was determined by the method of LOWRY et al. 11. The enzyme assays conditions l~ were the following: o.6/,mole NADH, i mg ferricytochrome c or I Fmole K3Fe(CN)6, o.71 mmole Tris-chloride (pH 8.5) and 4O-lOO Fg of protein. The final reaction volume was 3.o ml. The assays were carried out in a Beckman DU spectrophotometer at 25 °. The reaction was started with the addition of NADH and absorbance at 55o m~ or 42o m~ was measured at 3o-sec intervals for a period of 15-2o rain. The non-enzymic reduction of the acceptor by NADH was determined each time and subtracted from the value obtained with the complete system. The millimolar extinction coefficients used were the following: NADH, 6.22 (340 m/~); ferricytochrome c, reduced m i n u s oxidized, 19.1 (55° m~); potassium ferricyanide, o.96 (42o m~). The specific activities were expressed as m~moles of acceptor reduced per min per mg protein. Intact or fragmented erythrocyte membranes catalyze the oxidation of NADH using cytochrome c as electron acceptor. Fig. I shows the time course of the reduction 0.38C
/
:& 0.360
E
,/
,/
© U~ 13
.?
*6 Q340 C
m
<~ 0.320
0.30C
//.,,/: A/
o/*
o/ 2/ 0
i 3
I 7
I
[
11 15 Time (rain)
Fig. i. T i m e course of N A D H : c y t o c h r o m e c o x i d o r e d u c t a s e a c t i v i t y c a t a l y z e d b y e r y t h r o c y t e ghost. A - - h , , i n t a c t ; O - - O , f r a g m e n t e d . T h e a s s a y conditions are described in t h e t e x t .
Biochim. Biophys. Acta, 12o (1966) 165-169
SHORT COMMUNICATIONS
167
of cytochrome c obtained with both preparations. The mean value of the specific activity of this reaction obtained from seven different preparations of intact membranes was 7.9 ± 0.72 mtzmoles/min per mg protein and the specific activity of washed fragmented membranes was of the same order of magnitude in four preparations; this activity varied between 3.76 and 7.69. These results indicate that the enzymic activity of our preparation is a particulate one and suggest that it is free of cytoplasmic NADH:(acceptor) oxidoreductase (EC 1.6.99.3). Intact membranes obtained from blood washed with dextran TM, with a diminished content of white cells (one leukocyte per ioooo erythrocytes) and platelets, show similar specific activities (9.2 4- 0.9)to that found ill the untreated blood. This finding shows that the enzymic activity present in these ghosts is associated with the red blood cell membrane. The pH-dependence curve of this NADH: cytochrome c oxidoreductase activity was determined. Maximal activities were observed between pH 7.5 and 8.5, with a marked decrease below pH 6.0 and above pH 9.0. The specificity of the electron transfer reactions of the erythrocyte ghosts was studied with several electron donors and electron acceptors (Table I). The high specificity towards NADH as compared with N A D P H underlines the most important difference between this particulate enzyme and the soluble N A D H : (acceptor) oxidoreductase present in red blood cell lysates 14. The high activity of the NADH:ferricyanide oxidoreductase (lO7 times higher than that of the NADH: cytochrome c oxidoreductase) displayed by the red-cell membrane is remarkable. This fact is considered15 to be a rather good measure of the turnover rate of the electron transport between NADH and flavoprotein, and it would suggest that the flavoprotein component is retained in the erythrocyte membrane. The red blood cell membrane preparation was essentially free of succinate- and lactate-oxidizing activities. Moreover, N A D P H was not oxidized at all, indicating that the ghosts were also free from NADPH:(acceptor) oxidoreductase (EC 1.6.99.1). Even though 2,6dichlorophenolindophenol was appreciably reduced, methylene blue was not reduced under these conditions. This result is further evidence that the enzymic activity of erythrocyte ghosts was not a residual catalytic activity of the soluble NADH:(acceptor) oxidoreductase of red-cell lysates 1~. In order to investigate the mechanism of NADH oxidation by erythrocyte TABLE I ELECTRON TRANSPORTACTIVITIESOF HUMANBRYTHROCYTEGHOSTS Experimental conditions were as described in the text. Activities are expressed as m#moles of acceptor reduced/min per mg of protein. Number of experiments is given in parentheses. Mean values ± S.E. Electron donors
NADH, 0.6 #mole NADPH, 0.6 #mole Succinate, 20/zmoles Lactate, 0.5 #mole
Electron acceptors Cytochrome c
Ferricyanide
(~ mg)
(I #mole)
2,fi-Dichlorophenolindophenol Methylene blue Oxygen (20 #g) (20 #g)
7.9 ± o.72 (13) None None None
568 ± 46 (24) None None None
17.5 (2) ----
None None ---
None None None None
Biochim. Biophys. Acta, 12o (1966) 165-169
168
SHORT COMMUNICATIONS
membranes, different inhibitors were used to s t u d y the N A D H : f e r r i c y a n i d e oxidoreductase activity. 2,4-Dinitrophenol a n d a n t i m y c i n did n o t inhibit this reaction, while it was entirely blocked b y IO ~ M p-chloromercuribenzoate. This result indicates t h a t in this electron transfer reaction - S H groups are critically involved. It is possible t h a t this group m a y be involved in the nucleotide interactions with erythrocyte m e m b r a n e s as it has been suggested for the microsomal N A D H : c y t o c h r o m e c oxidoreductase (ref. i0). On the other hand, VANSTEVENINCK17 has shown recently the presence of - S H group on the outer surface of the erythrocyte ghost. I t has been postulated t h a t the presence of - S H groups is related to the structural integrity of the cell surface Is. Our finding suggests t h a t at least a fraction of t h e m is related to the electron transfer reaction t a k i n g place on the plasma membrane. Since the ferricyanide reduction in erythrocyte ghosts is insensitive to a n t i m y c i n it m a y proceed b y only one pathway, p r o b a b l y at the dehydrogenase level. I n consequence the N A D H : f e r r i c y a n i d e oxidoreductase a c t i v i t y would be a measure of the t u r n o v e r rate of the N A D H enzyme. However, the possibility t h a t other intermediates in the m e m b r a n e could be involved in the reduction of ferricyanide c a n n o t be elimin a t e d m. The present investigation shows t h a t red-cell m e m b r a n e s have a N A D H : (acceptor) oxidoreductase activity. This reaction proceeds with cytochrome c, ferricyanide a n d 2,6-dichlorophenolindophenol as electron acceptors, b u t it is not clear from these experiments t h a t all these electron transfer reactions proceed b y only one pathway. The possibility t h a t the reduction of cytochrome c could proceed through a n intermediate b o u n d to the m e m b r a n e is u n d e r active investigation. Of particular interest is the relationship t h a t m a y exist in this type of m e m b r a n e between biochemical organization a n d t r a n s p o r t processes. Our results have shown t h a t u n d e r conditions of s t r u c t u r a l integrity of red blood cell ghosts, electron transfer reactions take place. However, it remains to be determined whether or not these reactions play a role u n d e r physiological conditions. This research was partially supported b y the U.S. Air Force u n d e r G r a n t A F - A F O S R 788-65 to M. CANESSA-FISCHER; monitored b y the Air Force Office of Scientific Research of the Office of Aerospace Research. Laboratorio de Biofisica, Facultad de Ciencias, Universidad de Chile, Santiago (Chile)
1 2 3 4 5 6 7 8 9 Io ii 12
[TALO ZAMUDIO MITZY CANESSA
P. MITCHELL,Biochem. Soc. Symp. Cambridge, Engl., 22 (1963) 142. P. MITCHELL,J. Gen. Microbiol., 29 (1962) 25. P. EMMELOTAND C. J. 130s, Biochim. Biophys. Acta, 58 (19621 374R. L. POST, C. R. MERRIT, C. R. KINSOLVINGAND C. D. ALBRIGHT, J. Biol. Chem., 235 (~96o) 1796. A. ASKARI, Science, 141 (z963) 44. S. L. SCHRIER,J. Clin. Invest., 42 (19631 756. M. M. OLIVEIRAAND M. VAUGHAN,J. Lipid Res., 5 (19641 156. S.H. RICHARDSON,H. O. HULTINAND D. E. GREEN, Proc. Natl. Acad. Sci. U.S., 5 ° (19631 821. T. OHNISHI,J. Biochem., 52 (19621 3o7 . D. DANON,A. NEVO AND Y. YIARIKOVSKI,Bull. Res. Council Israel, Sect. E, 6 (1956) 36. O. H. LowRY, N. J. ROSEBROUGH,A. L. FARRAND t~. J. RANDALL,J. Biol. Chem., 193 (19511 265. H. I{. MAHLER, in S. P. COLOWICKAND N. Q). KAPLAN, Methods in Enzymology, Part II, New York, 1955, p. 688.
Biochim. Biophys. Acta, 12o (1966) 165-169
169
SHORT COMMUNICATIONS 13 14 15 16 17 I8 19
P. A. MARKS, A. GELLHORN AND C. KIDSON, J. Biol. Chem., 235 (196o) 2579. E. M. SCOTT AND J. C. MCGRAW, J. Biol. Chem., 237 (1962) 249. Y. HATEFI, A. G. HAAVIK AND P. JURTSHUK, Biochim. Biophys. Acta, 52 (1961) lO6. P. STRITTMATTER, J. Biol. Chem., 234 (1959) 2661. J. VANSTEVENINCK, R. I. WEED AND A. ROTHSTEIN, J. Gen. Physiol., 48 (1965) 617. R. E. BENESCH AND R_. BENESCH, Arch. Biochem., 48 (1954) 38. S. MINAKAMI, R. L. RINGLER AND T. SINGER, J. Biol. Chem., 237 (1962) 569 .
Received November 9th, 1965 Biochim. Biophys. Acta, 12o (1966) 165-169
BBA
43 o75
Fluorescence spectra of human serum albumin in the pH region of the N - F transition Bovine serum albumin undergoes a marked structural change in the region near p H 4 (ref. i). AOKI AND FOSTER2, studying the electrophoretic changes occurring in bovine serum albumin at low ionic strength in this p H region, coined the term " N - F transition" to describe the transformation of bovine serum albumin from the normal state (N) to a form having a faster rate of migration (F). Evidence from ultraviolet difference spectra suggests that acidification of bovine serum albumin solutions results in a perturbation of the environment of tyrosyl residues, probably at least in part by a general conformational change identifiable with the N - F transitiona, 4. H u m a n serum albumin is very similar to bovine serum albumin in physical parameters I and also undergoes an N - F transition near p H 4 (ref. 5). Since changes in the degree of tyrosyl--carboxylate hydrogen bonding and unfolding of the albumin molecule are postulated to occur in the N - F transition, it was of interest to examine the fluorescence spectrum of human serum albumin at low p H to see if changes in tyrosine and t r y p t o p h a n emission occurred. H u m a n serum albumin (Pentex Corp., Kankakee, Ill. ; Lot No. 9) was dialyzed against distilled water. Solutions were adjusted to various p H values with HC1 and made up to an ionic strength of 0.02 with NaC1. A Beckman expanded-scale meter was used to measure p H against a p H 4.0 potassium phthalate standard. A calibrated Aminco-Bowman spectrophotofluorimeter was employed to measure the spectra as described previously. For the present work, the emission monochromator was fitted with a grating blazed for m a x i m u m transmission at 300 m/z. With an RCA IP28 photomultiplier tube, the photodetector system gave all essentially flat response (relative quanta vs. wavelength) over the region 280-400 m/z as determined b y the calibration method of MELHUISH~. The spectra shown m a y therefore be considered "true" emission spectra. The solutions were excited at 275 m/~ through a horizontally oriented polarizing filter in order to minimize scattered lightS; no filters or polarizers were placed ill the emitted beam. Representative spectra are shown in Fig. i, which is divided into three sections so that the individual curves m a y be better visualized. As the p H is lowered, there is a continuous change in the shape of the emission curve. The fluorescence m a x i m u m changes as follows: p H 5.42 and 5.05, 335 m/~: p H 4.42, 331 m~; p H 3.75, 324 m ~ ; Biochim. Biophys. Acta, 12o (1966) 169-171
SHORT COMMUNICATIONS
kidney 12, rabbit brain 13, at low concentration, and at higher concentrations in individual experiments in embryo chicken heart 14. Several explanations for these phenomena exist. We have earlier put forward a hypothesis based upon supposition of a conformational effect of ouabain upon the enzyme itself relative to a metal-binding site z°. This would suppose that ouabain affects the enzyme conformation in a manner which the pre-reaction conformation of the protein disposes. In consequence, availability of the binding site for potassium might be made greater or less by a single action of ouabain. This thesis may be economically applied to the present observations. This work was supported in part by U.S. Public Health Service Research Grant H E 09 571; in part by a Grant-in-Aid from the American Heart Association.
Biochemistry Department, University of Texas Medical Branch, Galveston, Texas (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13 14
HARRY DARROW BROWN
A. SCHWARTZ, Biochem. Biophys. Res. Commun., 9 (1962) 3Ol. J. C. SKOU, Biochim. Biophys. Acta, 23 (1957) 394J. C. SKOU, Physiol. Rev., 45 (1965) 596. T. Z. CS~KY, Rev. Physiol,, 27 (1965) 415 . K. AH~aED AND J. D. JUDAH, Canad. J. Biochem., 43 (1965) 877. M. V. RILEY, Exptl. Eye Res., 3 (1964) 76. A. SCHWARTZ AND A. H. LASETER, Biochem. Pharmacol., 13 (1964) 337. A. ASKARI AND J. C. FRATANTONI, Biochim. Biophys. Acta, 71 (1963) 229. L. E. HOKIN AND D. REASA,. Biochim. Biophys. Acta, 9o (1964) 176. H. D. BROWN, ~h~. J. 2~-EUCERE, A. M. ALTSCHUL AND W. J. EVANS, Life Sci., (1965) 1439. K. S. LEE AND D. H. YU, Biochem. Pharmacol., 12 (1963) 1253. R. F. PALMER AND B. R. NECHAY,J. Pharmacol. Exptl. Therap., 146 (1964) 92. S. L. BUNTING, N. M. HAWKINS AND M. R. CANADY,Biochem. Pharmacol., 13 (1964) 13. R. L. KLEIN, Biochim. Biophys. Acta, 73 (1963) 488.
Received September 27th, 1965 Revised manuscript received November i2th, 1965 Biochim. Biophys. Acta, 12o (1966) 162-165
BBA 43 077
Nicotinamide-adenine dinucleotide dehydrogenase activity of human erythrocyte membranes Evidence accumulated in the last few years supports the concept that the cell membrane is a metabolically active structure, where a "vectorial" assembly of enzymes may be closely related with transport processes 1. A number of enzymic activities has been found in membranes of bacteria and liver cells2,3. In red blood cell "ghosts", (Na+-K+-MgZ+)-activated ATP phosphohydrolase (EC 3.6.1.3) (ref. 4), AMP aminohydrolase (EC 3.5.4.6) (ref. 5), D-glyceraldehyde-3-ph°sphate:NAD oxidoreducta~e (phosphorylating)-ATP:D-glyceraldehyde 3-phosphotransferase complex (EC 1.2.1.12 and 2.7.I.28), together with a triple enzyme sequence of pentose phosphate pathway*, have been described. Also, incorporation of fatty acids into phospholipids has been detected in erythrocyte ghosts 7. Other findings seem to indicate that the red-cell membrane has, as a feature in common with other biological membranes, Biochim. Biophys. Acta, 12o (1966) 165-169
166
SHORT COMMUNICATIONS
a structural 8 and a contractile protein 9. Electron transfer reactions have been shown to proceed only in mitochondria and microsomal membrane. We were interested in investigating the mammalian cell membrane for the occurrence of enzymes of electron transfer reactions that presumably could play a role as energy devices in the plasmalemma. In this work we report evidence that red-cell membranes are able to oxidize NADH using different electron acceptors. Red blood cell ghosts were prepared from fresh venous blood of normal human adults according to DANON, NEVO AND MARIKOVSKI1° o r POST e¢ al. 4. The membranes obtained by DAXON'S procedure had a white color (99-95 % of the hemoglobin had been removed) and they appeared to be intact under the phase-contrast microscope; when the technique of POST was used they were shown to be fragmented. Red blood cell ghosts were kept in o.I M histidine-imidazole buffer (pH 7.1) at 4 ° for a period not longer than 4 days. Protein was determined by the method of LOWRY et al. 11. The enzyme assays conditions l~ were the following: o.6/,mole NADH, i mg ferricytochrome c or I Fmole K3Fe(CN)6, o.71 mmole Tris-chloride (pH 8.5) and 4O-lOO Fg of protein. The final reaction volume was 3.o ml. The assays were carried out in a Beckman DU spectrophotometer at 25 °. The reaction was started with the addition of NADH and absorbance at 55o m~ or 42o m~ was measured at 3o-sec intervals for a period of 15-2o rain. The non-enzymic reduction of the acceptor by NADH was determined each time and subtracted from the value obtained with the complete system. The millimolar extinction coefficients used were the following: NADH, 6.22 (340 m/~); ferricytochrome c, reduced m i n u s oxidized, 19.1 (55° m~); potassium ferricyanide, o.96 (42o m~). The specific activities were expressed as m~moles of acceptor reduced per min per mg protein. Intact or fragmented erythrocyte membranes catalyze the oxidation of NADH using cytochrome c as electron acceptor. Fig. I shows the time course of the reduction 0.38C
/
:& 0.360
E
,/
,/
© U~ 13
.?
*6 Q340 C
m
<~ 0.320
0.30C
//.,,/: A/
o/*
o/ 2/ 0
i 3
I 7
I
[
11 15 Time (rain)
Fig. i. T i m e course of N A D H : c y t o c h r o m e c o x i d o r e d u c t a s e a c t i v i t y c a t a l y z e d b y e r y t h r o c y t e ghost. A - - h , , i n t a c t ; O - - O , f r a g m e n t e d . T h e a s s a y conditions are described in t h e t e x t .
Biochim. Biophys. Acta, 12o (1966) 165-169
SHORT COMMUNICATIONS
167
of cytochrome c obtained with both preparations. The mean value of the specific activity of this reaction obtained from seven different preparations of intact membranes was 7.9 ± 0.72 mtzmoles/min per mg protein and the specific activity of washed fragmented membranes was of the same order of magnitude in four preparations; this activity varied between 3.76 and 7.69. These results indicate that the enzymic activity of our preparation is a particulate one and suggest that it is free of cytoplasmic NADH:(acceptor) oxidoreductase (EC 1.6.99.3). Intact membranes obtained from blood washed with dextran TM, with a diminished content of white cells (one leukocyte per ioooo erythrocytes) and platelets, show similar specific activities (9.2 4- 0.9)to that found ill the untreated blood. This finding shows that the enzymic activity present in these ghosts is associated with the red blood cell membrane. The pH-dependence curve of this NADH: cytochrome c oxidoreductase activity was determined. Maximal activities were observed between pH 7.5 and 8.5, with a marked decrease below pH 6.0 and above pH 9.0. The specificity of the electron transfer reactions of the erythrocyte ghosts was studied with several electron donors and electron acceptors (Table I). The high specificity towards NADH as compared with N A D P H underlines the most important difference between this particulate enzyme and the soluble N A D H : (acceptor) oxidoreductase present in red blood cell lysates 14. The high activity of the NADH:ferricyanide oxidoreductase (lO7 times higher than that of the NADH: cytochrome c oxidoreductase) displayed by the red-cell membrane is remarkable. This fact is considered15 to be a rather good measure of the turnover rate of the electron transport between NADH and flavoprotein, and it would suggest that the flavoprotein component is retained in the erythrocyte membrane. The red blood cell membrane preparation was essentially free of succinate- and lactate-oxidizing activities. Moreover, N A D P H was not oxidized at all, indicating that the ghosts were also free from NADPH:(acceptor) oxidoreductase (EC 1.6.99.1). Even though 2,6dichlorophenolindophenol was appreciably reduced, methylene blue was not reduced under these conditions. This result is further evidence that the enzymic activity of erythrocyte ghosts was not a residual catalytic activity of the soluble NADH:(acceptor) oxidoreductase of red-cell lysates 1~. In order to investigate the mechanism of NADH oxidation by erythrocyte TABLE I ELECTRON TRANSPORTACTIVITIESOF HUMANBRYTHROCYTEGHOSTS Experimental conditions were as described in the text. Activities are expressed as m#moles of acceptor reduced/min per mg of protein. Number of experiments is given in parentheses. Mean values ± S.E. Electron donors
NADH, 0.6 #mole NADPH, 0.6 #mole Succinate, 20/zmoles Lactate, 0.5 #mole
Electron acceptors Cytochrome c
Ferricyanide
(~ mg)
(I #mole)
2,fi-Dichlorophenolindophenol Methylene blue Oxygen (20 #g) (20 #g)
7.9 ± o.72 (13) None None None
568 ± 46 (24) None None None
17.5 (2) ----
None None ---
None None None None
Biochim. Biophys. Acta, 12o (1966) 165-169
168
SHORT COMMUNICATIONS
membranes, different inhibitors were used to s t u d y the N A D H : f e r r i c y a n i d e oxidoreductase activity. 2,4-Dinitrophenol a n d a n t i m y c i n did n o t inhibit this reaction, while it was entirely blocked b y IO ~ M p-chloromercuribenzoate. This result indicates t h a t in this electron transfer reaction - S H groups are critically involved. It is possible t h a t this group m a y be involved in the nucleotide interactions with erythrocyte m e m b r a n e s as it has been suggested for the microsomal N A D H : c y t o c h r o m e c oxidoreductase (ref. i0). On the other hand, VANSTEVENINCK17 has shown recently the presence of - S H group on the outer surface of the erythrocyte ghost. I t has been postulated t h a t the presence of - S H groups is related to the structural integrity of the cell surface Is. Our finding suggests t h a t at least a fraction of t h e m is related to the electron transfer reaction t a k i n g place on the plasma membrane. Since the ferricyanide reduction in erythrocyte ghosts is insensitive to a n t i m y c i n it m a y proceed b y only one pathway, p r o b a b l y at the dehydrogenase level. I n consequence the N A D H : f e r r i c y a n i d e oxidoreductase a c t i v i t y would be a measure of the t u r n o v e r rate of the N A D H enzyme. However, the possibility t h a t other intermediates in the m e m b r a n e could be involved in the reduction of ferricyanide c a n n o t be elimin a t e d m. The present investigation shows t h a t red-cell m e m b r a n e s have a N A D H : (acceptor) oxidoreductase activity. This reaction proceeds with cytochrome c, ferricyanide a n d 2,6-dichlorophenolindophenol as electron acceptors, b u t it is not clear from these experiments t h a t all these electron transfer reactions proceed b y only one pathway. The possibility t h a t the reduction of cytochrome c could proceed through a n intermediate b o u n d to the m e m b r a n e is u n d e r active investigation. Of particular interest is the relationship t h a t m a y exist in this type of m e m b r a n e between biochemical organization a n d t r a n s p o r t processes. Our results have shown t h a t u n d e r conditions of s t r u c t u r a l integrity of red blood cell ghosts, electron transfer reactions take place. However, it remains to be determined whether or not these reactions play a role u n d e r physiological conditions. This research was partially supported b y the U.S. Air Force u n d e r G r a n t A F - A F O S R 788-65 to M. CANESSA-FISCHER; monitored b y the Air Force Office of Scientific Research of the Office of Aerospace Research. Laboratorio de Biofisica, Facultad de Ciencias, Universidad de Chile, Santiago (Chile)
1 2 3 4 5 6 7 8 9 Io ii 12
[TALO ZAMUDIO MITZY CANESSA
P. MITCHELL,Biochem. Soc. Symp. Cambridge, Engl., 22 (1963) 142. P. MITCHELL,J. Gen. Microbiol., 29 (1962) 25. P. EMMELOTAND C. J. 130s, Biochim. Biophys. Acta, 58 (19621 374R. L. POST, C. R. MERRIT, C. R. KINSOLVINGAND C. D. ALBRIGHT, J. Biol. Chem., 235 (~96o) 1796. A. ASKARI, Science, 141 (z963) 44. S. L. SCHRIER,J. Clin. Invest., 42 (19631 756. M. M. OLIVEIRAAND M. VAUGHAN,J. Lipid Res., 5 (19641 156. S.H. RICHARDSON,H. O. HULTINAND D. E. GREEN, Proc. Natl. Acad. Sci. U.S., 5 ° (19631 821. T. OHNISHI,J. Biochem., 52 (19621 3o7 . D. DANON,A. NEVO AND Y. YIARIKOVSKI,Bull. Res. Council Israel, Sect. E, 6 (1956) 36. O. H. LowRY, N. J. ROSEBROUGH,A. L. FARRAND t~. J. RANDALL,J. Biol. Chem., 193 (19511 265. H. I{. MAHLER, in S. P. COLOWICKAND N. Q). KAPLAN, Methods in Enzymology, Part II, New York, 1955, p. 688.
Biochim. Biophys. Acta, 12o (1966) 165-169
169
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P. A. MARKS, A. GELLHORN AND C. KIDSON, J. Biol. Chem., 235 (196o) 2579. E. M. SCOTT AND J. C. MCGRAW, J. Biol. Chem., 237 (1962) 249. Y. HATEFI, A. G. HAAVIK AND P. JURTSHUK, Biochim. Biophys. Acta, 52 (1961) lO6. P. STRITTMATTER, J. Biol. Chem., 234 (1959) 2661. J. VANSTEVENINCK, R. I. WEED AND A. ROTHSTEIN, J. Gen. Physiol., 48 (1965) 617. R. E. BENESCH AND R_. BENESCH, Arch. Biochem., 48 (1954) 38. S. MINAKAMI, R. L. RINGLER AND T. SINGER, J. Biol. Chem., 237 (1962) 569 .
Received November 9th, 1965 Biochim. Biophys. Acta, 12o (1966) 165-169
BBA
43 o75
Fluorescence spectra of human serum albumin in the pH region of the N - F transition Bovine serum albumin undergoes a marked structural change in the region near p H 4 (ref. i). AOKI AND FOSTER2, studying the electrophoretic changes occurring in bovine serum albumin at low ionic strength in this p H region, coined the term " N - F transition" to describe the transformation of bovine serum albumin from the normal state (N) to a form having a faster rate of migration (F). Evidence from ultraviolet difference spectra suggests that acidification of bovine serum albumin solutions results in a perturbation of the environment of tyrosyl residues, probably at least in part by a general conformational change identifiable with the N - F transitiona, 4. H u m a n serum albumin is very similar to bovine serum albumin in physical parameters I and also undergoes an N - F transition near p H 4 (ref. 5). Since changes in the degree of tyrosyl--carboxylate hydrogen bonding and unfolding of the albumin molecule are postulated to occur in the N - F transition, it was of interest to examine the fluorescence spectrum of human serum albumin at low p H to see if changes in tyrosine and t r y p t o p h a n emission occurred. H u m a n serum albumin (Pentex Corp., Kankakee, Ill. ; Lot No. 9) was dialyzed against distilled water. Solutions were adjusted to various p H values with HC1 and made up to an ionic strength of 0.02 with NaC1. A Beckman expanded-scale meter was used to measure p H against a p H 4.0 potassium phthalate standard. A calibrated Aminco-Bowman spectrophotofluorimeter was employed to measure the spectra as described previously. For the present work, the emission monochromator was fitted with a grating blazed for m a x i m u m transmission at 300 m/z. With an RCA IP28 photomultiplier tube, the photodetector system gave all essentially flat response (relative quanta vs. wavelength) over the region 280-400 m/z as determined b y the calibration method of MELHUISH~. The spectra shown m a y therefore be considered "true" emission spectra. The solutions were excited at 275 m/~ through a horizontally oriented polarizing filter in order to minimize scattered lightS; no filters or polarizers were placed ill the emitted beam. Representative spectra are shown in Fig. i, which is divided into three sections so that the individual curves m a y be better visualized. As the p H is lowered, there is a continuous change in the shape of the emission curve. The fluorescence m a x i m u m changes as follows: p H 5.42 and 5.05, 335 m/~: p H 4.42, 331 m~; p H 3.75, 324 m ~ ; Biochim. Biophys. Acta, 12o (1966) 169-171