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Built-in co-operativity of oxygen binding by arthropod hemocyanins
J. Mol. Biol. (1977) 115, 257-261
Built-in Co-operativity
of Oxygen Binding by Arthropod Hemocyanins
Leirus puin Oxygen binding by artlwopod Ilemocyauil~ fro111 t,tlc: scorpion yctestriatus and the crabs Telphltsa jlwviatilzs and Oc,ypoda cwwr was studied iu c’a2+, Mg2+-free solutions. The binding was found to be co-operative in all three cases. Our results and a re-examinat,ion of the litc~ratnre lead IIS to conclude that, co-operative oxygen binding is a built-in featuw common to artllropod hemocyanins, distinguishing them from molllwz hcmoc>~anitls lvhrre co-oprrativity is cv~l~ditional 11por1 the presence of ("a2+ or Mg2+.
Hemocganin is a copper-containing rcS~Jira~~Ory prot)t:in found in the hemolymph of molluscs and arthropods. Renewed interest in htmocyanin cent’ers on the oxygen binding function of the protein. and the way in which calcium and magnesium ions normally found in the hemolymph affect’ the binding behavior. In the presence of Ca” + or Mg2 + , oxygen binding by hemocyanin has been found to be co-operative (Ghirrtt’i, 1962 ; Van Holde $ Van Bruggen, 1971). In the absence of t,hese ions, non-co-operative binding has bcaen observed (Ghirctti, 1962; Van Holde & Van Hruggen, 1971). though some rsceptions (e.g. Pick&t rt al.. 1966) were reported. As H consequence, it has become generally accepted that’ Ca2 + and Mg2 + bring ahout co-opcrativity (Van Holde & Van Hruggrn, 197 1). Recently. we began a study of the oxygen binding properties of arthropod hcmocya,nin from the scorpion Leirus rluilbrl/*“s~rintus. We found a marked co-operative I)(havior in calcium-free solut’ions. Sincac such a behavior, if est8ablished. would rrpr(asttnt a fundarnental departure from current aoncept,s regarding the mechanism of c( I-opcrativit\’ in hemocyanins, I\.(’ decidtd t’o extend our study to hemocyanins from ot’her sources. Our findings, and a re-examination of the literature, lead us to the conclusion that co-operative oxygen lkding. in the absence of Ca2+ and Mg2+, is a huilt-in feature common to hemocbyanins from members of the arthropod pl~ylum. di4npuishin,g t,hern from molluscan hemocyanins. Hcmocyanin from L. quinquwfriatus was prepared a,s previously described (Klarman rt nl.. 1977). Hrmolymph from t,he freshwater crab T~l~husa~uviatilds and the short: cral) Ocgpoda cur,sor was collected by means of a ltypodermio syringe from the base of a Lvnlking leg and set aside to clot at room t’empcraturc. The clot was squeezed and the liquid was separated on filter paper. The tilt&e was centrifuged for 20 minut,es at 27,000g in a Sorvall preparabive centrifuge. The supernatant, was subjected to further centrifugation for three hours at 226,000 g, whereupon hemocyanin sedirnent c,d. The pellet was dissolved in 0.1 &I-Tris.HCl buffer (pH 7.3). All thrte hcmooyanins were dialyzed against, buffer containing lo- 3 hz-EDTA in order to remove Ca’ + and Mg2.+. The EDTA was subsequent~ly removed by exhaust8ive dialysis against, t’he desired buffer. Examination by atomic ‘absorption. using a \‘arian model AAS, showed less than 10m6 M-C~~+or Mg2+. Oxygen binding \vas measured spectrophot,ometrica,lly using a Cary 118 C spectrophot#ometer, by following the 310 nm absorption band, in a tonometer as described elsewhere (Er-cl et al., 1972). Increments of air or oxygen at one atmosphere were added, the: choice depending upon t,he oxygen
258
affinity of hemocyanin performed at 20°C.
A. KLARMAN
-4N1)
at the experimental
11:. IIANIEEJ,
conditions.
All oxygen
t~it~rations ww
Figure 1 presents oxygen binding data for L. quinquestriatus hrmocpwnin in t,he absence of Ca2+ and Mg2+ at pH 8.5. Under these condit)ions, t,his hemocyanin exists as whole molecules, szO,W= 37 S. The saturation curve, fractional saturatiou P versus partial oxygen pressure 21, is seen bo be sigmoidal, and the Hill plot,. log P/(1 - P) tIersus log p, is of the type expected for co-operative binding (Wyman. 1964), with slopes n = 1 at P = 0 and P = 1, and n > 1 at’ intermediate values of P. The maximal value of the slope, nmax = 6.8, is among the highest reported for hemocyanins. Co-operative behavior in the absence of Ca2+ and Mg2+ was also obtained in the oxygen titration of hemocyanins from T. jhviatilis and 0. cursoT (Fig. I). A comparison of the oxygen binding behavior of native and sbripped (Ca2 + . hemocvanin as well as hemocyanin containing conMg 2+-free) L. quinquestriatus range, is presented in centrations of Ca2 + , and Ca2 + plus Mg2+, in the physiological Figure 2. Our results definitely show that, in some arthropods a,t least, hemocyanin can bind oxygen co-operatively in the absence of Ca2+ and Mg2+. In the light’ of these findings, it seemed justifiable to question whether the generally accepted view conditioning co-operativity on the presence of these ions is equally valid for bot’h molluscan and arthropod hemocyanins. Examination of the literature indeed showed that
FIQ. 1. Hill plots of oxygen equilibrium for L. quinquestriatus (a), T.$uviatiZis (b), and 0. CUTSD~ at pH 8.5, 7.3 and 7.5, (c) hemocyanins in the absence of Ca2+ and Mg2+, in 0.1 M-Tris.HCl, respectively, at 20°C. Asymptotes with a slope of unity were drawn. The data for L. puinpuestriatus hemocyanin are also presented in the form of a saturation curve in the insert,.
LETTERS
TO
THE
EDITOR
“59
0.6
I 0.5
I.0 ILog
PQ2
Pm. 2. Oxygen titration curvw for L. qminyrlc,stricctus hc~mocyanin in tho abwnce (0) and the lwewncr of 1O-2 a&a2+ (A), 5 n’ 1W3 ~r-Caz+ plus 5x IO-” wMg2+ (A), in 0.1 nc-Tris.HCl (pH 8.5), 20°C. The curve for the hPmolymph containing 10 2 ~(‘a”+ and 4x 1O-3 nr-Mg2+ (pH 8.5 and 20°C) is also shown (0).
stud& where removal of Ca2 + and Mg 2 + brought’ about’ loss of co-operativity involved hemocyanin from molluscan sources (Stedman & Stedman, 1928; Konings et al., 1969; Er-el et nl., 1972; Vannoppen-Ver Eecke & Lontie. 1973; Wood et al., 1977). However, the experimental data obtained on arthropod hemocyanins do not warrant’> in our opinion, viewing co-operativity of oxygen binding to be conditional on the presence of these ions. The early observations of Pickett et al. (1966) on the cooperative oxygen binding of hemocyanin from the lobster Homarus americanus in Ca2+-free solution have already been mentioned. Binding dat’a from more recent studies do not indicate loss of co-operat’ivity upon removal of Ca2 + and Mg2+, as is borne out by the values exceeding unity obtained for the slope of the Hill plot (Table 1). The value nmax -= 1 obtained for Limulus polyphemus hemocyanin, the only entry not exceeding unity in Table 1, is not really indicative of an exceptional behavior, being a necessary consequence of the fact that this hemocyanin, upon treatment with EDTA to remove Ca,2+ and Mg 2+ ~ dissociates into monomeric units carrying one oxygen binding site each (Sullivan et al., 1974). Significantly a small but definite co-operative effect, ?A,,, =: 1.28, was found by Miller & Van Holde (1974) in the course of a very careful stud!l of t’hc oxygen binding properties of CalZianas,~a californipnsie hemocyanint. On the basis of our results as well as the ones wc were able to find in the literabure, we conclude that co-operativit’y is a built-in feature of oxygen binding by arthropod hemocyanins. This means that’ the conformational transition which lies behind the manifestation of co-operative behavior is an inherent property of the molecule and not) brought about by the addition of an external t#ectjor. This is in contrast to the situation in molluscan hemocyanins where co-operativity is generated by the presence of Ca2+ or Mg2 +. In arthropod hemocyanins, the effect of Ca2 + and Mg2 + seems restricted to modifying the affinity and the degree of co-operativity (Fig. 2) in a way reminiscent of the role played by diphosphoglycerate in the hemoglobin-oxygen system. t The smell value of nmax may have to do with the divsoclation (39 S --z 17 S) of C. cnlifomiensis homocyanin upon removal of C’az+ and Mg2+ (Roxby et nl., 1974).
260
Merostomata Tachypleus tridentatus l%mulus polyphemus Limulus polyphemus
Estimated fiwn Fig. 2 of Hwang & I’rmg (I 970) Redficltl (1930) Sullivan et tel. (1974)
3 1.0 I.0
c!rustacea Procnmbarus simulans Homarus nmericnnus Carcinus mediterruneus Potamon edulis ~d’iU?W8SC, cnlzfornien.sis &j@2OdU
2.6
Larimer I% Riggs (1964) Pick&t, et rtl. (1966) Fig. 4 of (‘hant,ler et rtl. (l!)i:l) Fig. 5 of chantlor et ccl. (1973) Miller & Van Holcle (1974) This work This wo~lc
4 6.8
Loewe & Linzm This work
2.1-4.1 3.6 P.O-f.fi I .3-4.0 1.28 24
CUTSOF
l’elphusn jluaiutilis Arachnida Cupiennius 8alei Leirus quinquestriatus
(1975)
Finally, it has long been realized that molluscan and arthropod hemocyanins are quite dissimilar structurally. In molluscan hemocyanins, there is one oxygen binding site per 50,000 daltons, while in arthropod hemocyanins one site is associated with 1966). Circular dichroic spectra suggest 75,000 daltons (Ghiretti-Magaldi et aZ., marked differences in the environment of the copper-oxygen site (Nickerson & Van Holde, 1971). Sedimentation (Eriksson-Quensel & Svedberg, 1936) and electron et al., 1966) microscopy (Levin, 1963 ; Van Bruggen et al., 1963; Fernandez-Moran reveal that the pattern of subunit assembly is entirely different in the two groups of proteins. One may wonder whether the differences in the mechanism of co-operativity discussed here are the functional expression on a molecular level of the long-noted structural differences between hemocyanins from t,he two phyla. Department of Biochemistry The George S. Wise Center for Life SciolIccs Tel-Aviv University, Tel-Aviv, Israel Received
17 May
ALEXANOEH. KLARS~AN EZRA DANIEL
1977 REFERENCES
Chantler, E. N., Harris, R. 11. $ Bannister, W. H. (1973). Comp. Biochem. Physiol. 46A, 333-343. Er-el, Z., Shaklai, N. & Daniel, E. (1972). J. Mol. Biol. 64, 341-352. Eriksson-Quensel, I.-B. & Svedberg, T. (1936). Biol. Bull. 71, 498-547. Fernandez-Moran, H., Van Bruggen, E. F. J. & Ohtsuki, M. (1966). J. Mol. Biol. 16, 191-207. Ghiretti, F. (1962). In Ozygenases (Hayaishi, O., ed.), pp. 517 -553, Academic Press, NC\% York. Ghiretti-Magaldi, A., Nuzzolo, C. & Ghiretti, I?. (1966). Biochemistry, 5, 1943-1951. Hwang, J. C. & Fung, C. P. (1970). Comp. Biochem. Physiol. 37, 573-579.
LETTERS Klarmitn, Konings,
TO THE
EDITOR
201
A., Shaklai, N. & Daniel, E. (1977). Rio&m. Biophys. Actu, 490, 322-330. W. N., Van Driel, R., Van Bruggerl, E. F’. .J. & Gruber. M. (1969). Riochim. Biophys. dcta, 194, 55-66. Larinwr, J. L. 6r Itiggs, A. I?. (1964). Camp. Biochem. l’hysiol. 13, 3&46. Lcvin, 0. (1963). d&iv Kemi, 21, l-13. LOCWY~, K. & L&en, B. (1975). J. Camp. I’hysid. 98, 147 156. Miller. K. & Van Holde, K. E. (1974). Biochemistry, 13, 1668--1674. Nickcrsoll, K. IV. 8: Van Holtlc, K. E. (1971). Camp. Riochem. Physiol. 39B, 855-872. Pick&t, S. M., Higgs, A. P. & Larimer, J. L. (1966). Sciertfz, 151, 1005-1007. Tietlfkltl, X. C. (1!430). Biob. Bull. 58, 238-255. Ibxby. Ii.. Miller. K., Blair, D. P. & Van Holcie. K. E. (1974). Hiochemistry, 13, 1662- 1668. Stdrnan. E. B Stdman, E. (192X). Bioch,em. J. 22, 889-901. Slllli\-ali, B., Huriwvent~urn, ,I. ‘s! l3otlav0rltllra. C’. ( 1974). I’m-. Std. .4catl. ,sci., I’.S.A. 71, 2.558 -2562. Vat) Hrugge~t, E. k’. .J., Scltuitt~tl. \‘., JVeihcnga, E. H. k (:rllhcr. M. (1963). J. Mol. Biol. 7. “3R 253. Vurl Holtl(a, Ii. E. & Van Bruggw, E. b’. J ( 197 1). 1 II Hioloyical Macromolecules Series (‘l‘imasileff, S. N. & Fnsmarr. (:. D., eds), vol. 5. part A, pp. I~-55, Marcel Dekker, rttc., NW York. Varllll,l’l)“‘-Ver Eeckc, Th. & Lontie, R. (1973). Camp. Biochem. E’hysiol. 45B, 945-954. Wood. E. .I ., Cayley, G. K. 8.zPearson, J. S. (1977). J. ,Ylol. Biol. 109, l- 1 I. W\‘yrnall. J-. (1961). Advan. Protein Chem. 19, 223.. 286.
Built-in Co-operativity
of Oxygen Binding by Arthropod Hemocyanins
Leirus puin Oxygen binding by artlwopod Ilemocyauil~ fro111 t,tlc: scorpion yctestriatus and the crabs Telphltsa jlwviatilzs and Oc,ypoda cwwr was studied iu c’a2+, Mg2+-free solutions. The binding was found to be co-operative in all three cases. Our results and a re-examinat,ion of the litc~ratnre lead IIS to conclude that, co-operative oxygen binding is a built-in featuw common to artllropod hemocyanins, distinguishing them from molllwz hcmoc>~anitls lvhrre co-oprrativity is cv~l~ditional 11por1 the presence of ("a2+ or Mg2+.
Hemocganin is a copper-containing rcS~Jira~~Ory prot)t:in found in the hemolymph of molluscs and arthropods. Renewed interest in htmocyanin cent’ers on the oxygen binding function of the protein. and the way in which calcium and magnesium ions normally found in the hemolymph affect’ the binding behavior. In the presence of Ca” + or Mg2 + , oxygen binding by hemocyanin has been found to be co-operative (Ghirrtt’i, 1962 ; Van Holde $ Van Bruggen, 1971). In the absence of t,hese ions, non-co-operative binding has bcaen observed (Ghirctti, 1962; Van Holde & Van Hruggen, 1971). though some rsceptions (e.g. Pick&t rt al.. 1966) were reported. As H consequence, it has become generally accepted that’ Ca2 + and Mg2 + bring ahout co-opcrativity (Van Holde & Van Hruggrn, 197 1). Recently. we began a study of the oxygen binding properties of arthropod hcmocya,nin from the scorpion Leirus rluilbrl/*“s~rintus. We found a marked co-operative I)(havior in calcium-free solut’ions. Sincac such a behavior, if est8ablished. would rrpr(asttnt a fundarnental departure from current aoncept,s regarding the mechanism of c( I-opcrativit\’ in hemocyanins, I\.(’ decidtd t’o extend our study to hemocyanins from ot’her sources. Our findings, and a re-examination of the literature, lead us to the conclusion that co-operative oxygen lkding. in the absence of Ca2+ and Mg2+, is a huilt-in feature common to hemocbyanins from members of the arthropod pl~ylum. di4npuishin,g t,hern from molluscan hemocyanins. Hcmocyanin from L. quinquwfriatus was prepared a,s previously described (Klarman rt nl.. 1977). Hrmolymph from t,he freshwater crab T~l~husa~uviatilds and the short: cral) Ocgpoda cur,sor was collected by means of a ltypodermio syringe from the base of a Lvnlking leg and set aside to clot at room t’empcraturc. The clot was squeezed and the liquid was separated on filter paper. The tilt&e was centrifuged for 20 minut,es at 27,000g in a Sorvall preparabive centrifuge. The supernatant, was subjected to further centrifugation for three hours at 226,000 g, whereupon hemocyanin sedirnent c,d. The pellet was dissolved in 0.1 &I-Tris.HCl buffer (pH 7.3). All thrte hcmooyanins were dialyzed against, buffer containing lo- 3 hz-EDTA in order to remove Ca’ + and Mg2.+. The EDTA was subsequent~ly removed by exhaust8ive dialysis against, t’he desired buffer. Examination by atomic ‘absorption. using a \‘arian model AAS, showed less than 10m6 M-C~~+or Mg2+. Oxygen binding \vas measured spectrophot,ometrica,lly using a Cary 118 C spectrophot#ometer, by following the 310 nm absorption band, in a tonometer as described elsewhere (Er-cl et al., 1972). Increments of air or oxygen at one atmosphere were added, the: choice depending upon t,he oxygen
258
affinity of hemocyanin performed at 20°C.
A. KLARMAN
-4N1)
at the experimental
11:. IIANIEEJ,
conditions.
All oxygen
t~it~rations ww
Figure 1 presents oxygen binding data for L. quinquestriatus hrmocpwnin in t,he absence of Ca2+ and Mg2+ at pH 8.5. Under these condit)ions, t,his hemocyanin exists as whole molecules, szO,W= 37 S. The saturation curve, fractional saturatiou P versus partial oxygen pressure 21, is seen bo be sigmoidal, and the Hill plot,. log P/(1 - P) tIersus log p, is of the type expected for co-operative binding (Wyman. 1964), with slopes n = 1 at P = 0 and P = 1, and n > 1 at’ intermediate values of P. The maximal value of the slope, nmax = 6.8, is among the highest reported for hemocyanins. Co-operative behavior in the absence of Ca2+ and Mg2+ was also obtained in the oxygen titration of hemocyanins from T. jhviatilis and 0. cursoT (Fig. I). A comparison of the oxygen binding behavior of native and sbripped (Ca2 + . hemocvanin as well as hemocyanin containing conMg 2+-free) L. quinquestriatus range, is presented in centrations of Ca2 + , and Ca2 + plus Mg2+, in the physiological Figure 2. Our results definitely show that, in some arthropods a,t least, hemocyanin can bind oxygen co-operatively in the absence of Ca2+ and Mg2+. In the light’ of these findings, it seemed justifiable to question whether the generally accepted view conditioning co-operativity on the presence of these ions is equally valid for bot’h molluscan and arthropod hemocyanins. Examination of the literature indeed showed that
FIQ. 1. Hill plots of oxygen equilibrium for L. quinquestriatus (a), T.$uviatiZis (b), and 0. CUTSD~ at pH 8.5, 7.3 and 7.5, (c) hemocyanins in the absence of Ca2+ and Mg2+, in 0.1 M-Tris.HCl, respectively, at 20°C. Asymptotes with a slope of unity were drawn. The data for L. puinpuestriatus hemocyanin are also presented in the form of a saturation curve in the insert,.
LETTERS
TO
THE
EDITOR
“59
0.6
I 0.5
I.0 ILog
PQ2
Pm. 2. Oxygen titration curvw for L. qminyrlc,stricctus hc~mocyanin in tho abwnce (0) and the lwewncr of 1O-2 a&a2+ (A), 5 n’ 1W3 ~r-Caz+ plus 5x IO-” wMg2+ (A), in 0.1 nc-Tris.HCl (pH 8.5), 20°C. The curve for the hPmolymph containing 10 2 ~(‘a”+ and 4x 1O-3 nr-Mg2+ (pH 8.5 and 20°C) is also shown (0).
stud& where removal of Ca2 + and Mg 2 + brought’ about’ loss of co-operativity involved hemocyanin from molluscan sources (Stedman & Stedman, 1928; Konings et al., 1969; Er-el et nl., 1972; Vannoppen-Ver Eecke & Lontie. 1973; Wood et al., 1977). However, the experimental data obtained on arthropod hemocyanins do not warrant’> in our opinion, viewing co-operativity of oxygen binding to be conditional on the presence of these ions. The early observations of Pickett et al. (1966) on the cooperative oxygen binding of hemocyanin from the lobster Homarus americanus in Ca2+-free solution have already been mentioned. Binding dat’a from more recent studies do not indicate loss of co-operat’ivity upon removal of Ca2 + and Mg2+, as is borne out by the values exceeding unity obtained for the slope of the Hill plot (Table 1). The value nmax -= 1 obtained for Limulus polyphemus hemocyanin, the only entry not exceeding unity in Table 1, is not really indicative of an exceptional behavior, being a necessary consequence of the fact that this hemocyanin, upon treatment with EDTA to remove Ca,2+ and Mg 2+ ~ dissociates into monomeric units carrying one oxygen binding site each (Sullivan et al., 1974). Significantly a small but definite co-operative effect, ?A,,, =: 1.28, was found by Miller & Van Holde (1974) in the course of a very careful stud!l of t’hc oxygen binding properties of CalZianas,~a californipnsie hemocyanint. On the basis of our results as well as the ones wc were able to find in the literabure, we conclude that co-operativit’y is a built-in feature of oxygen binding by arthropod hemocyanins. This means that’ the conformational transition which lies behind the manifestation of co-operative behavior is an inherent property of the molecule and not) brought about by the addition of an external t#ectjor. This is in contrast to the situation in molluscan hemocyanins where co-operativity is generated by the presence of Ca2+ or Mg2 +. In arthropod hemocyanins, the effect of Ca2 + and Mg2 + seems restricted to modifying the affinity and the degree of co-operativity (Fig. 2) in a way reminiscent of the role played by diphosphoglycerate in the hemoglobin-oxygen system. t The smell value of nmax may have to do with the divsoclation (39 S --z 17 S) of C. cnlifomiensis homocyanin upon removal of C’az+ and Mg2+ (Roxby et nl., 1974).
260
Merostomata Tachypleus tridentatus l%mulus polyphemus Limulus polyphemus
Estimated fiwn Fig. 2 of Hwang & I’rmg (I 970) Redficltl (1930) Sullivan et tel. (1974)
3 1.0 I.0
c!rustacea Procnmbarus simulans Homarus nmericnnus Carcinus mediterruneus Potamon edulis ~d’iU?W8SC, cnlzfornien.sis &j@2OdU
2.6
Larimer I% Riggs (1964) Pick&t, et rtl. (1966) Fig. 4 of (‘hant,ler et rtl. (l!)i:l) Fig. 5 of chantlor et ccl. (1973) Miller & Van Holcle (1974) This work This wo~lc
4 6.8
Loewe & Linzm This work
2.1-4.1 3.6 P.O-f.fi I .3-4.0 1.28 24
CUTSOF
l’elphusn jluaiutilis Arachnida Cupiennius 8alei Leirus quinquestriatus
(1975)
Finally, it has long been realized that molluscan and arthropod hemocyanins are quite dissimilar structurally. In molluscan hemocyanins, there is one oxygen binding site per 50,000 daltons, while in arthropod hemocyanins one site is associated with 1966). Circular dichroic spectra suggest 75,000 daltons (Ghiretti-Magaldi et aZ., marked differences in the environment of the copper-oxygen site (Nickerson & Van Holde, 1971). Sedimentation (Eriksson-Quensel & Svedberg, 1936) and electron et al., 1966) microscopy (Levin, 1963 ; Van Bruggen et al., 1963; Fernandez-Moran reveal that the pattern of subunit assembly is entirely different in the two groups of proteins. One may wonder whether the differences in the mechanism of co-operativity discussed here are the functional expression on a molecular level of the long-noted structural differences between hemocyanins from t,he two phyla. Department of Biochemistry The George S. Wise Center for Life SciolIccs Tel-Aviv University, Tel-Aviv, Israel Received
17 May
ALEXANOEH. KLARS~AN EZRA DANIEL
1977 REFERENCES
Chantler, E. N., Harris, R. 11. $ Bannister, W. H. (1973). Comp. Biochem. Physiol. 46A, 333-343. Er-el, Z., Shaklai, N. & Daniel, E. (1972). J. Mol. Biol. 64, 341-352. Eriksson-Quensel, I.-B. & Svedberg, T. (1936). Biol. Bull. 71, 498-547. Fernandez-Moran, H., Van Bruggen, E. F. J. & Ohtsuki, M. (1966). J. Mol. Biol. 16, 191-207. Ghiretti, F. (1962). In Ozygenases (Hayaishi, O., ed.), pp. 517 -553, Academic Press, NC\% York. Ghiretti-Magaldi, A., Nuzzolo, C. & Ghiretti, I?. (1966). Biochemistry, 5, 1943-1951. Hwang, J. C. & Fung, C. P. (1970). Comp. Biochem. Physiol. 37, 573-579.
LETTERS Klarmitn, Konings,
TO THE
EDITOR
201
A., Shaklai, N. & Daniel, E. (1977). Rio&m. Biophys. Actu, 490, 322-330. W. N., Van Driel, R., Van Bruggerl, E. F’. .J. & Gruber. M. (1969). Riochim. Biophys. dcta, 194, 55-66. Larinwr, J. L. 6r Itiggs, A. I?. (1964). Camp. Biochem. l’hysiol. 13, 3&46. Lcvin, 0. (1963). d&iv Kemi, 21, l-13. LOCWY~, K. & L&en, B. (1975). J. Camp. I’hysid. 98, 147 156. Miller. K. & Van Holde, K. E. (1974). Biochemistry, 13, 1668--1674. Nickcrsoll, K. IV. 8: Van Holtlc, K. E. (1971). Camp. Riochem. Physiol. 39B, 855-872. Pick&t, S. M., Higgs, A. P. & Larimer, J. L. (1966). Sciertfz, 151, 1005-1007. Tietlfkltl, X. C. (1!430). Biob. Bull. 58, 238-255. Ibxby. Ii.. Miller. K., Blair, D. P. & Van Holcie. K. E. (1974). Hiochemistry, 13, 1662- 1668. Stdrnan. E. B Stdman, E. (192X). Bioch,em. J. 22, 889-901. Slllli\-ali, B., Huriwvent~urn, ,I. ‘s! l3otlav0rltllra. C’. ( 1974). I’m-. Std. .4catl. ,sci., I’.S.A. 71, 2.558 -2562. Vat) Hrugge~t, E. k’. .J., Scltuitt~tl. \‘., JVeihcnga, E. H. k (:rllhcr. M. (1963). J. Mol. Biol. 7. “3R 253. Vurl Holtl(a, Ii. E. & Van Bruggw, E. b’. J ( 197 1). 1 II Hioloyical Macromolecules Series (‘l‘imasileff, S. N. & Fnsmarr. (:. D., eds), vol. 5. part A, pp. I~-55, Marcel Dekker, rttc., NW York. Varllll,l’l)“‘-Ver Eeckc, Th. & Lontie, R. (1973). Camp. Biochem. E’hysiol. 45B, 945-954. Wood. E. .I ., Cayley, G. K. 8.zPearson, J. S. (1977). J. ,Ylol. Biol. 109, l- 1 I. W\‘yrnall. J-. (1961). Advan. Protein Chem. 19, 223.. 286.