Kinetic parameters need clarifying

514 side reactions are excluded. Carraway et al.7.8, for example, stressed the fact that in aqueous solutions at acidic and neutral pH carbodiimides react to almost the same extent with carboxyls as they do with sulfhydryls, so that modification of carboxyl groups alone requires additional precautions. As a consequence, the use of SH-reagents to protect sulfhydryl groups from possible interaction with carbodiimide was recommended (SH-groups may be regenerated afterwards) 8. Under similar conditions tyrosine 8 and serine 9 residues may be modified by carbodiimides. All this suggests that the mode of action of DCCD in a given system should not be postulated to involve a certain carboxylic group, unless directly demonstrated. (2) Solioz proposes 1 that whenever the proton translocating activity of an enzyme is found to be inhibited by DCCD and, in addition, (14C)DCCD labelling of a certain subunit is observed, a H+-pump should be postulated. The situation is slightly more complex. In the mitochondrial b-c a complex, for instance, DCCD seems to bind to cytochrome b and/or subunit VIII and to inhibit proton translocation by the enzyme; at the same time it appears to induce other effects like intermolecular cross-linking between two or three subunits ~° and decreased antimycin binding it. In such a complicated system a careful study of the time-course of all phenomena observed has eventually helped to identify the one event responsible for the inhibition; this is the crosslinking of subunits V and VII and not the binding of DCCD ~°. Moreover, DCCD is also likely to induce undetectable intramolecular cross-linking within one of the subunits of the studied enzyme and the possibility that this causes the observed inhibition cannot usually be excluded. Mitochondrial b- G complex may also be used as an example of a system that interacts with DCCD and is inhibited by it, but probably translocates protons in a way different from the F~Fo-ATPase. The principle of a proton-motive Q-cycle operating in the b-G complex has not been confuted or even seriously questioned till now. On the contrary, the experimental support for it is growing ~2. (3) Solioz himself1 cites examples which do not fit in with his view of DCCD as a probe for proton-translocating enzymes: such as bacteriorhodopsin, a proton pump which is neither labelled nor inhibited by D C C D and Ca :+ATPase of the sarcoplasmic reticulum, an enzyme which is not involved in proton transport but labelled and inhibited

T I B S - D e c e m b e r 1984

by DCCD. He neglects them, however, in his conclusion. The list of examples can be extended: the interaction of DCCD with the isolated photosynthetic centres from R h o d o s p i r i l l u m r u b r u m or with the mitochondrial anion-transporting pore. All this points to a broad reactivity of DCCD towards different enzymatic systems rather than to its supposed specificity for H÷-pumps. We do not intend to criticize the general use of DCCD in studies of enzymes of bioenergetic relevance. This reagent, although broadly reactive, has in some cases provided valuable scientific information. We cannot, however, agree with Solioz that the binding and the inhibition by D C C D which have been demonstrated in some cation transporting proteins should be used as a diagnostic tool for them. In a recent review on this subject 13 we stress a general warning that the syllogism 'If it has rained the streets are wet' cannot be reversed to say 'Since the streets are wet it has rained'. One of the reviewers positively commented that for some scientists: 'Sometimes even wet streets are raining'...

References 1 Sofioz, M. (1984) Trends Biochem. Sci. 9, 309312 2 Sebald, W. and Watchter, E. (1978) in Energy Conservation in Biological Membranes (Schiller, G. and Klingenberg, M., eds),

Springer-Vedag 3 Prochaska, L. J., Bisson, R., Capaldi, R. A., Steffens, G. C. M. and Buse, G. (1981) Biochim. Biophys. Aeta 637, 360-373 4 Casey,R. P., Thelen, M. and Azzi,A. (1980)J. Biol. Chem. 255, 3994-4000 5 Murphy, A. J. (1981) J. Biol. Chem. 256, 12046-12050 6 Kurzer, F. and Duraghi-Zadeh, K. (1%7) Chem. Rev. 67, 107-152 7 Carraway, K. L. and Triplett, R. B. (1970) Biochim. Biophys. Acta 200, 564-566 8 Carraway, K. L. and Koshland, D. E. Jr (1971) Meth. Enzymol. 26, 616-623 9 Banks, T. E., Blossey,B. K. and Shafer, J. A. (1%9) J. Biol. Chem. 244, 6323-6333 10 Natqcz,M. J., Casey, R. P. and Azzi,A. (1983) Biochim. Biophys. Acta 724, 75-82 11 Clejan, L. and Beattie, D. S. (1983) J. Biol. Chem. 258, 14271-14275 12 Rich, P. R. (1984) Biochim. Biophys. Acta 768, 53-79 13 Azzi,A., Casey, R. P. and Natqcz,M. J. (t984) Biochim. Biophys. Acta 768, 209-226 ANGELO AZZI and MACIEJ J. NA-LI~CZ Medizinisch-Chemisches Institut der Universit~it Bern, Biihlstrasse 28, 3000 Bern 9, Switzerland.

Kinetic parameters need clarifying Mato and his associates ~ have recently concentrated (~- 200x) purified phosphatidylethanolamine methyltransferase (PMTase) from rat liver microsomes: this preparation contains two major proteins whose molecular weights are 50 000 and 25 000, respectively. The 25 000 protein is photoaffinity-labeled with 8-azido-S-adenosylmethionine (AdoMet), whereas the 50 000 protein is phosphorylated by cyclic AMP-dependent kinase with the concomitant increase in PMTase activity; thus, they proposed that the 25 000 protein is the catalytic subunit, whereas the 50 000 protein is the regulatory subunit of PMTase. Since other preparations of PMTase purified to a similar extent from the s a m e source 2.3, can convert phosphatidylethanolamine (PtdEth) to phosphatidylcholine (PtdCho), they stated that only one enzyme catalyses the three-step methylation of PtdEth to form PtdCho 4. PtdEth

S-AdoMet ~ E1

The question how many enzymes are involved in this reaction has been debated since the discovery of this enzyme activity5. The reaction proceeds as shown in equation 1: Assuming that one step of this reaction does not affect the other steps, the formula postulated can be applied to determine the kinetic parameters; in the conditions which they describe as optimal, the Vm~x of E~, E 2 and E 3 a r e 175, 170 and 150 pmol mg -~ min -t, respectively6. The exogenous addition of monomethyl- and dimethyl-PtdEth, but not of PtdEth, results in an increase of these rates as follows: 175,250 and 600 pmol mg -t min t, respectively. Since all steps require S-AdoMet for activity, the concentration of S-AdoMet also affects the rate of each step. The substrate velocity curve of PMTase often has an inflection point around 1 bI.M and gives two apparent K,, values of 0.8 and 60 IxM, respectively 7. The K m for S-AdoMet

monomethyl-PtdEth

dimethyl-PtdEth E2

S-AdoMet PtdCho Equation 1

E3

515

TIBS - December 1984

S-AdoMet in the presence of exogenous monomethyl- and dimethyl-PtdEth are both about 60 p,M. As the concentration of S-AdoMet in the reaction decreases, the ratio of the amount of monomethylPtdEth accumulated relative to that of dimcthyl-PtdEth and PtdCho formed increases. These results suggest that lower concentrations of S-AdoMet shift the rate limiting step from E t to E 2 and/or E 3, and that the kinetic properties are E t are masked by those of E 2 (and E3). Furthermore, exogenous monomethyl-PtdEth is converted mainly to dimethyl-PtdEth (not to PtdCho), suggesting that the reaction does not proceed sequentially but rather randomly depending upon the availability of monoethyl- or dimethyl-PtdEth even in the conditions where PtdEth is saturated. Digestion of resealed vesicles or erythrocyte ghosts with trypsin from the inside or outside differentially inactivates E~ or E 2 (and E3)s,L The inactivation of E, and E 3 occurs in parallel. These observations suggested that the enzyme (PMTase I) which catalyses E 1is distinct from the enzyme(s) (PMTase II) which proceeds via E 2 and E 3. The involvement of two distinct enzymes, PMTase I and PMTase II, in the reaction has also been demonstrated by the genetic studies using Neurospora, Saccharomyces and rat basophilic leukemia cells l~n. Arguments are focused on the possibility that a point mutation in the same gene can modify the catalytic properties of PMTase to different phospholipid substrates. Similarly it can be argued that a partial digestion and denaturation of the single-enzyme molecule (during isolation) results in expression of different kinetic patterns with different substrates. Osawa and his associates have recently purified (1500×) PMTase from lymphocyte plasma membranes and physically separated PMTase I-like activity from PMTase II-like activity (personal communications). These preparations still contain phospholipase A 2 and other enzyme activities. Although rat liver microsomes contain high PMTase activity, it is surprising that PMTase occupies nearly 0.5% of the total membrane proteins as suggested by the finding that a 200-fold purification results in a homogeneous enzyme activity. Although the direct evidence for the catalytic and regulatory functions of 25 000 and 50 000 proteins as PMTase has not been shown, Mato et al. should clarify the complicated kinetic parameters of the single PMTase molecule (by modifications with proteases, phospholipid

substrates and S-AdoMet as described above) by studying the PMTase consisting of 25 000 catalytic subunit and 50 000 regulatory subunit. Saccharomyces has recently been shown to have the gene for PMTase I distinct from the gene for PMTase lI~k If the proposal of Mato et al. is correct then a novel phenomenon in developmental biology can occur: the transformation of two genes in the lower classes of eukaryocytes to one gene in the higher classes of eukaryocytes.

References 1 Verela, I., Merida, 1., Pajares, M. A., Vinalba, M. and Mato, J. (1984) Biochem. Biophys. Res. Commun. 122, 1065-1071 2 Tanaka, Y., Doi, O. and Akamatsu, Y. (1979) Biochem. Biophys. Res. Commun. 87, 11091115 3 Schneider, W. J. and Vance, D. (1979) J. Biol. Chem. 254, 3887-3891 4 Mato, J. M., Pajares, M. A. and Varela, 1.

(1984) Trends Biochem. Sci. 9, 471-472 5 Bremer, J. and Greenberg, D. M. (1961) Biochim. Biophys. Acta 43, 477-488 6 Audubert, F. and Vance, D. E. (1983) J. Biol. Chem. 258, 10695-10701 7 Sastry, R., Statham, J., Axelrod, J. and Hirata. F. (1981) Arch. Biophys. Biochern. 211,762769 8 Hirata, F. and Axelrod, J. (1978) Proc. Natl Acad. Sci. USA 75, 2348-2352 9 Higgins, J. A. (1981) Biochim. Biophys. Acta 640, 1-15 10 Scarborough, G. A. and Nyc, J. F. (1967) J. Biol. Chem. 254, 3886-3891 i1 Yamashita. S., Oshina, A., Nikawa, J. and Hosaka, K. (1982) Eur. J. Biochem. 128, 589595 12 McGiveney, A., Crews, F. T., Hirata, F., Axelrod, J. and Siraganian, R. P. (1981) Proc. Natl Acad. Sci. USA 78, 6176--6180 FUSAO HIRATA

Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, USA.

Reply from J. M. Mato As mentioned by Hirata in the above Letter to the Editor, based on experiments where erythrocyte ghosts I or resealed vesicles from rat liver microsomes 2 are incubated with trypsin, it has been concluded that mammalian cells have two different phospholipid methyltransferases and that both enzymes are localized at different sides of the membranes. However, Audubert and Vance 3 after digestion with trypsin of sealed rat liver microsomes could not confirm these results. These authors concluded that all phospholipid methyltransferase activities are on the external side of the vesicles. The conclusion about two methyltransferases in mammalian cells has also been based on the requirement of Mg 2+ for the conversion of PtdEth to PtdEthMet but not for the conversion of PtdEthMet to PtdChot. However this dependence on Mg 2+ has not been confirmed by other authors 5-v. It is our opinion that, based only on published data, it is not possible to conclude that in mammalian cells two methyltransferases are

necessary to convert PtdEth to PtdCho. Finally, we have never claimed to have a homogeneous phospholipid methyltransferase but only to have partially purified this enzyme activity, as it is mentioned in the title of Ref. 1 in the above Letter to the Editor by Hirata.

References l Hirata, F. and Axelrod, J. (1978) Proc. Natl Acad. Sci. USA 75, 2348-2352 2 Higginns, J. A. (1981) Biochim. Biophys. Acta 640, 1-15 3 Audubert, F. and Vance, D. E. (1984) Biochim. Biophys. Acta 792, 359-362 4 Hirata, F. and Axelrod, J. (1980) Science 209, 1082-1090 5 Prasad, C. and Edwards, R. M. (1981) J. Biol. Chem. 256, 130L~-13003 6 Schneider, W. J. and Vance, D. E. (1979) J. Biol. Chem. 254, 3886-3891 7 Hoffman, D. R. and Cornatzer, W. E. (1981) Lipids 16, 53.3-540 J. M. MATO

Fundaci6n Jim6nez Diaz, Avd. Reyes Catolicos 2, Ciudad Universitaria, Madrid 280GO, Spain.

Phosphofructokinase and glycolytic f l u x There are so many points to question in the 'Open Question' for September entitled 'Is phosphofructokinase the rate-limiting step of glycolysis? 1, that for brevity I can only mention a few: (1) Whilst the authors may have chosen their title with rhetorical (or even ironic) intent, it was unwise in an article purporting to use the concepts of the distribution of flux developed by Kacser and Burns 2 and Heinrich and Rapoport 3. 'How much does phospho-

fructokinase contribute to the control of glycolytic flux?' would have been more acceptable. (2) As was mentioned briefly in their summary, the question has been answered for human erythrocytes (a 'Group I' cell in the article) by Heinrich, Rapoport and co-workers using a theoretical approach 4, the results of which were in line with their experimental studies 5. The flux control coefficient6 (or sensitivity) of phosphofructokinase was