Knowledge about ATPases ignored

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Letters Interactions of glycolytic enzymes A number of the statements in the article entitled 'A reappraisal of the binding of cytosolic enzymes to erythrocyte membranes' by Maretzki, Reimann and Rapoport ~invite me to take issue. In this article, data are presented on the influence of ionic strength on binding studies for glycolytic enzymes in human erythrocytes, and from this very limited base several sweeping statements are made in relation to the reality of enzyme interactions with cell structure in general. As an example of one of the conclusions, the authors state that evidence for the existence of significant membrane binding of intact enzymes in erythrocytes is doubtful under physiological conditions - a deduction which is largely based on a comparison of the extent of lhe binding of these enzymes under hypotonic and isotonic conditions, the diminished binding in the latter situation leading to the above conclusion. Unfortunately, in their experiments, the authors have failed to recognize or examine one of the most characteristic and influential factors in the establishment of a realistic physiological status. It is widely recognized, for example, that the cellular milieu is characterized by a high concentration of protein, and it is known that molecular crowding by macromolecules in this environment exerts a strong influence on the degree of interactions between soluble enzymes and particulate matter in the cell. Yet the authors carried out their extractions with protein-free solutions in all reported cases. Despite the emphasis in this paper on the importance of physiological conditions in such experiments, then, it must be said that the data presented do not adequately represent the cellular situation, and indeed does little more than reproduce a fact well known to workers in this field for some time - thai hypotonicity increases the extent of enzyme binding in dilute aqueous (i.e. nonphysiological) solutions 2,3. On this rather insubstantial base, then, the authors move on to much broader conclusions. They make the statements that assumptioas of the existence of complexes of glycolytic enzymes in other cell types have been based largely on work with erythro-

cytes, that there is a lack of experimental basis for the binding of glycolytic enzymes in general, and that proponents of these multienzyme complexes generally neglect to recognize the significance of physiological conditions in their experiments. In defense of the work of many colleagues, I would challenge these statements, and would contend that the arguments ignore much of the substantial bank of data which has been built up over the last 20 years, utilizing a variety of techniques and attesting to the reality of binding of glycolytic enzymes under physiological conditions in a wide variety of tissues 2 7. My concern in this matter would be that the significance of such extensive and painstaking work may be downgraded by too ready a credence based on the speculation in Ref. 1. I would agree with the authors that details of the spatial relationships and metabolic interactions of these adsorbed multienzyme

complexes remain to be clarified in many instances, but in view of the evidence available in the literature, I cannot accept that the binding of cytosolic enzymes to cellular structure has not been demonstrated to be a physiologically relevant phenomenon. References I Maretzki, D., Reimann, B. and Rapoport, S. M. (1989) Trends Biochem. Sci. 14, 93-96 2 Masters, C. J. (1978) Trends" Biochem. Sci. 3, 20(~208 3 Masters, C. J. (1981) C R C Crit. Rev. Biochem. 11, 1/15-143 4 Srere, P. A. (1987)Annu. Rev. Biochem. 56, 89-124 5 Clegg, J. S. (1984) Bioessays 1,129 131 60v~idi, J. (1989) Trends Biochem. Sci. 13, 48(v~t90 7 Harris, S. and Winzor, D. (1987) Biochim. Biophys. Acta 911,121-126

COLIN MASTERS

Griffith University, Nathan, Brisbane, Queensland, Australia 4111.

Reply from Rapoport Nowhere in our article I have we made a statement denying the general possibility of enzyme interactions with cell structure; we emphasized this view in the last sentence. As indicated by the title, we restricted our survey to erythrocytes. Masters does not provide any new evidence for membrane binding of glycolytic enzymes or for metabolic chaneling. The only point that deserves further comment is the influence of high protein concentrations on enzyme binding. This possibility was excluded in the experiments of Rich et al. ~ (Ref. 25 in our article). Furthermore, no changes of kinetic parameters of

G A P D H in intact cells were demonstrated by Brindle et al. 3 (Ref. 30 in our article). References 1 Maretzki, D., Reimann, B. and Rapoport, S. M. (1989) Trends Biochem. Sci. 14, 93-96 2 Rich, G. T., Pryor, J. S. and Dawson, A. P. (1985) Biochern. Biophys. Acta 817, 61~6 3 Brindle, K. M., Campbell, I. D. and Simpson, R. J. (1982) Biochem. J. 2/18,583 592

S.M. RAPOPORT

Institute of Biochemistry, Faculty of Medicine (Charit6), Humboldt University, 1040 Berlin, G D R .

Knowledge about ATPases ignored Silver et al., in their otherwise excellent review on bacterial resistance ATPases 1, present a model for the cadmium efflux ATPase (Fig. 4 of their review) which is derived from the model of animal Ca2+-ATPase proposed by Brandl et al. 2 Some features of this model, however, have not been sup-

ported by subsequent experimental evidence. The evidence for the so called 'transduction domain' was based on uncoupling between ATP hydrolysis and Ca 2+ transport after tryptic cleavage within this domain. More recent studies have failed to find uncoupling after

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this tryptic cleavage 3. On the other hand, site-directed mutagenesis of conserved residues within this domain in the Saccharomyces cerevisiae ATPase suggest that it contains the catalytic site for the hydrolysis of the phosphorylated intermediate4. Mapping of a vanadate-resistant mutant of Schizosaccharomyces pombe ATPase within this domain 5 is also consistent with a function in phosphatase catalysis. The so-called 'hinge' domain was postulated by analogy to dehydrogenases to allow for movement of ATP-binding and phosphorylation domains6. Chemical modification of Na +, K+-ATPase by ATP derivatives7"8 and site-directed mutagenesis of Saccharomyces ATPase a, however, clearly indicate that this region is part of the ATP-binding site. Therefore, the transduction domain should be renamed as the phosphatase domain and the hinge domain should be included within the ATP-binding or kinase domain 4. Thus, Fig. 4 of Silver et al. 1 should be viewed with caution because it highlights features of the

model which are not supported by experimental evidence. References 1 Silver, S., Nucifora, G., Chu, L. and Misra, T. K. (1989) Trends Biochem. Sci. 14, 76-80 2 Brandl, C. ,I., Green, N. M., Korczak, B. and MacLennan, D. H. (1986) Ce1144, 597-607 3 Torok, K., Trinnaman, B. J. and Green, N. M. (1988) Eur. J. Biochem. 173,361-367 4 Portillo, F. and Serrano, R. (1988) E M B O J . 7, 1793-1798 5 Ulaszewski, S., Van Herck, J. C., Dufour, J. P., Kulpa, J., Niewenhuis, B. and Goffeau, A. (1987) J. Biol. Chem. 262,223-228 6 MacLennan, D. H., Brandl, C. J., Korczak, B. and Green, N. M. (1985) Nature 316, 696-700 7 0 t h a , T., Nagano, K. and Yoshida, M. (1986) Proc. Natl Acad. Sci. USA 83, 2(171-2075 8 Ovchinnikov, Y. A., Dzhandzugazyan, K. N., Lutscnko, S. V., Mustayev, A. A. and Modyanov, N. N. (1987) FEBS Lett. 217, 111-116 RAMON SERRANO

European Molecular Biology Laboratory, Meyerhofstrasse l, 6900 Heidelberg, FRG.

Purification of the D2 dopamine receptor In a recent edition of TIBS a short article described the cloning of the D2 dopamine receptor I. This is a major advance in the understanding of this receptor which is important not only as a site of drug action (anti-parkinsonian and anti-schizophrenic drugs) but also because of the reported alterations in D2 dopamine receptor number in schizophrenia. It is of further interest, therefore, that virtually simultaneously with the report of the cloning2, three reports appeared of the purification of the De dopamine receptor 3-5. Mr values of 92000 (Ref. 3) and 95000 (Ref. 4) for the receptor from brain, and 120000 for the receptor from anterior pituitary5 were reported. Whether the 9200095000 and 120000 species are proteolytically related forms of the same protein, or whether they represent distinct receptor subtypes is of some interest and remains to be determined. The cloning of the gene for the D2 dopamine receptor is of importance for a number of reasons including allowing the determination of the basis of alterations of receptor synthesis in pathological conditions and the relevance of such alterations to the disease state. However, a full understanding of the

receptor and in particular how it binds specifically with certain drugs and not others will require a variety of molecular biological, protein chemistry and biophysical studies based on a combination of the methods for gene cloning and receptor isolation.

Reply from Silver etaL We thank Dr Serrano for pointing out additional experimental data that suggest alternative functions for two of the conserved regions in the E1E2 ATPases. Experimental testing must be the basis for a model. If the first polypeptide region can be identified with phosphatase activity, that will make the model more useful. Whether the second specified region functions as a 'hinge' or is part of the ATP-binding region, it nevertheless is the most highly conserved region of this class of polypeptides. SIMON SILVER GUISEPPINA NUCIFORA LIEN CHU TAPAN K. MISRA

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60680, USA.

3 Elazar, Z., Kanety, H., David, C. and Fuchs, S. (1988) Biochem. Biophys. Res. Commun. 156,602~09 4 Williamson, R. A., Worrall, S., Chazot, P. L. and Strange, P. G. (1988) E M B O J. 7, 4129-4133 5 Senogles, S. E., Amlaiky, N., Falardeau, P. and Carom M. G. (1988) J. Biol. Chem. 263, 18996--19002

PHILIP STRANGE

References 1 Maelicke, A. (1989) Trends Biochem. Sci. 14, 41-42 2 Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C. A., Neve, K. A. and Civelli, O. (1988) Nature 336,783-787

Biological Laboratory, The University, Canterbury, Kent CT2 7N J, UK.

Glutamine and protein turnover In his Journal Club article in the January issue of TIBS 1, Watford discusses whether glutamine regulates skeletal muscle protein turnover. On the basis of Refs 2-4, he states that 'in rats the rate of skeletal muscle protein synthesis shows a good correlation with intracellular glutamine concentration'. Recently it has also been suggested5 that glutamine may inhibit protein degradation. The critical questions posed are (1) whether increased glutamine

concentration is a causative agent in promoting protein synthesis and in inhibiting degradation, or whether changes in the various parameters are merely coincidental, and (2) if the answer to (1) is the former, what mechanism(s) can be invoked. The rat diaphragm undergoes a pronounced but transient hypertrophy in the first few days following unilateral nerve section 6. Subsequently the tissue atrophies. In the early stages of the