The origin of biphasic Arrhenius plots of rat liver plasma-membrane 5'-nucleotidase [proceedings].

Intrinsic membrane proteins, which may only be solubilized by procedures that disrupt membrane structure, must all have domains that interact with their non-polar environment. The degree of interaction, however, varies from a strict requirement of phospholipid for activity (e.g. Ca2+-activated adenosine triphosphatase; Warren et al., 1974) t o a requirement for structural support only (e.g. cytochrome b5; Spatz & Strittmatter, 1971). One of the criteria for a membrane-bound enzyme that interacts with its lipid environment has been a biphasic Arrhenius plot. This is interpreted as a perturbation of the activation energy of the enzyme by a change in the physical structure of the lipid molecules in the membrane bilayer. There are, however, several other possibilities (Dixon & Webb, 1958; Silvius et al., 1978; Wynn-Williams, 1976), of which the principal alternative is that the membrane protein itself undergoes a conformational change at the break temperature which modifies its activation energy. This view has been supported by Kreiner et al. (1973) on the basis of the high cholesterol content of mammalian plasma membranes, which might be expected to damp out any rapid phase changes of membrane phospholipids. It has also been noted by Houslay et al. (1976) that the discontinuities in their e.s.r. data from plasma membranes were dependent on the presence of membrane protein and were not observed in vesicles made from lipids extracted from the same membranes but without protein. Even with Ca2+-activated adenosine triphosphatase, reconstitution of activity in phospholipid-free detergent micelles has been achieved and biphasic Arrhenius plots obtained similar to those for sarcoplasmic reticulum (Dean & Tanford, 1977; Tanford, 1978). S’-Nucleotidase may be defined as a n intrinsic membrane protein by its lack of solubility except in high detergent concentrations. The fact that it is a n ectoenzyme (DePierre & Karnovsky, 1974; Newby et al., 1975) synthesized on the rough endoplasmic reticulum (Bergeron et al., 1975) might also suggest that it is a transmembrane protein (Rothman & Lenard, 1977). Since the enzyme may be purified as a complex with sphingomyelin (Widnell, 1975) and also in a phospholipid-free form (Evans & Curd, 1973; Slavik et al., 1977), it provides an example where the relationship between Arrhenius plot and lipid environment may be directly tested. Fig. 1 shows the Arrhenius plot of the sphingomyelin complex of 5’-nucleotidase compared with partially purified forms of theenzyme containing no measurable phospholipid (less than 0.4mol of phospholipid/mol of protein, based on a protein mol.wt. of 140000). Not only was biphasicity retained in all preparations, but the break temperatures and activation energies were indistinguishable. The cholesterol content of the sphingomyelin-enzyme complex was similar t o that of plasma membranes on a protein basis, and no discontinuity over the temperature range 2 0 4 0 ° C was observed in the e.s.r. data obtained from the spin label 5-doxylstearic acid when incorporated into this material. The S’-nucleotidase activity, however, shows a 50 % change in activation energy at 30°C.