Reaction Characteristics and Stability of a Membrane-Bound Enzyme Reconstituted in Bilayers of Liposomes Azadeh Kheirolomoom, Shigeo Katoh,* Eizo Sada, and Ken-ichi Yoshida zyxw Chemical Engineering Department, Kyoto University, Kyoto 606, Japan Received May 31, 1990Accepted October 71, 1990 zyxwvut The effects ensuing from the interaction between mem- brane-bound sarcosine dehydrogenase and the surrounding lipids as well as the effects of membrane fluidity were de- scribed in this study. A 25-fold activation was observed upon the reconstitution of the enzyme in bilayers of SUVs made of zyxwvutsrqpo DMPC. The considerable decrease in zyxwvuts K, and in- crease in V,, suggest the induction of favorable conforma- tional changes in both the substrate-binding site and the catalytic site of the enzyme due to the lipid-protein interac- tion. In SUVs of negatively charged phospholipids, the en- zyme retained its initial activity over 1 month. The break point in the Arrhenius plot of the activity of reconstituted enzyme was found at temperatures close to the gel-liquid crystallinetransition point of the phospholipid showing that the activity is sensitive to the physical state of membrane zyxwvu phospholipids. Further, immobilization of the reconstituted enzyme by use of ENT prepolymer resulted in a high activ- ity, whereas no remarkable activity was detected with the immobilized enzyme without reconstitution. Key words: Membrane-bound enzyme Reconstitution Liposome Immobilization INTRODUCTION Almost all knowledge of enzyme mechanisms, immobi- lized techniques, and bioreactor designs for enzymatic reactions comes from the study of water-soluble en- zymes which act on water-soluble substrates! However, living cells possess highly specialized membranes in which many important biological reactions are mediated by the conjugated multi-membrane-bound enzymes.18 For example, prostaglandins, pharmacologicalIy active compounds, are synthesized via several membrane- bound enzymes.'6217 Yet, reports concerning the reaction mechanism and immobilization of these enzymes are very few and thus raise new and challenging questions in the enzyme engineering field. Cellular membranes are now accepted to be dynamic assemblies mainly of proteins and phospholipid^?*^ The nature of this organization requires that isolated puri- fied membrane-bound enzymes must be reconstituted into an appropriate environment before studying their structures, functions, and applications in the engineer- ing field.3,9,'5 Since natural membranes have very com- plicated structures, much more simple model systems such as liposomes [small unilamellar vesicles (SUVs)] * To whom all correspondence should be addressed. interacting with purified membrane-bound enzymes are commonly utilized>,13 Membrane-bound sarcosine dehydrogenase was se- lected in this study. Sarcosine dehydrogenase purified from zyxwvu Pseudomonas putidu is composed of four subunit monomers, each having a molecular weight of about 45,000 daltons. Sarcosine dehydrogenasewith creatinine amidohydrolase and creatine amidinohydrolase is in- volved in the metabolic pathway of creatinine to sarco- sine via creatine and further to glycine. This enzyme is useful for the clinical assay of creatinine, creatine and sarcosine contents in human serum and urine by cou- pling with the related two This membrane- bound enzyme was reconstituted in bilayers of SUVs of several phospholipids to investigate the effects of lipid- protein interactions and membrane fluidity changes on the enzyme activity and also the reaction character- istic of immobilized enzyme reconstituted in a lipo- philic environment. MATERIALS AND METHODS Materials Purified sarcosine dehydrogenase from Pseudomonas putida was purchased from Toyobo Co. Ltd. The following reagents were supplied by Sigma Chemical Co. : L-a- zyx dimyristoylphosphatidylcholine (DMPC), DL-a-dipalmitoylphosphatidylcholine (DPPC), DL-a-dipalmitoylphosphatidylethanolamine (DPPE), L-a-dimyristoylphosphatidyl-DL-glycerol (DMPG), L-a-dimyristoylphosphatidic acid (DMPA), L-a-phos- phatidyl-L-serin (PS) from bovine brain, nitro blue tet- razolium (NTB). Sarcosine, sodium cholate, Triton X-100, and phenazine methosulfate (PMS) were from Nacalai Tesque. Yolk phospholipid was purchased from The Green Cross Co. Photo-crosslinkable prepolymer ENT-4000 prepared from hydroxyethylacrylate, iso- phoron diisocyanate, and poly(ethy1ene glycol) was from Kansai Paint Co. Determination of Sarcosine Dehydrogenase Activity The activity of sarcosine dehydrogenase was assayed by coupling the enzyme reaction with a PMS-NTB system, Biotechnologyand Bioengineering, Vol. 37 Pp. 809-813 (1991) zyxwvuts 0 1991 John Wiley & Sons, Inc. CCC 0006-3592/91/090809-05$0400