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Abstract
D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
D-&Hydroxybutyrate dehydrogenase is a lipid-re- whereas the enzyme, inserted into phospholipid vesiquiring enzyme which is localized on the innerface of cles, is protected againsptroteolytic digestion suggestthe mitochondrial inner membrane
The effect of proteolytic digestion of D-P-hydroxybutyrate dehydrogenase in three different environments has been investigated 1)in submitochondrialvesicles, its native environment; 2) as the purified apodehydroganase in aqueous buffer and devoid of phospholipids; and 3) the purified active enzyme reconstituted into phospholipid vesicles
Protection against proteolysis is afforded by insertion of the enzyme into phospholipid vesicles; with trypsin, thermolysin, and S. aureus protease, essentially complete protection against proteolysis and inactivation is obtained
Summary
Vation of both apodehydrogenase and the enzyme-phosphopttdwwctamdgfwhherphrioehiaigceeeeTTondtotgitsehrrnhdaecnirrmmemdstoscheeedntaieyanihooaagertcoaidlyiittllwnesegnaommyyddstsalinssatoeriutee(iiiipnotdhroFnlnnonnihgfaonfsittaaogce(ptlenbttobFenst.ihheo,oipfatasi1vwfoehgbthdes(Dhes).eyeFoinpsipp,.tn-r2ilocLthuPhgiwaeo)erlpro.-otnyc.eioiirihtspflptoCdfft2yhiiysihereepv)podNeodrvpo.dtiasrmesgrhedAftDIttiotirserinnedpeaDxt-oiySscso,ccePascynep+etolstrt-abeaasriiea.nhtpvsuvtbihsnbytinhetilaoedloodytey,eiynfoolbrrnlofioaoprsriefnstoxeecnoelresa(forsoysgprFbvuscbotatviteSislciuhfgplhlitddu.eitrs.eooaeatystcbdfycauasri1eaayetaetuartnpi)uhhettslhvrPzeooliryeDlaieynyoAedetudn.umybieoacrsoGgdhstodbopeareawyppEgsimtpsrigaderriniotoipnootiinretnoihndolttteandtaeneeoegssoolttaahroeeieimloessnmynydbffweeapirdaitateott,cuairsirhnohcotgrtmoeooeidh-e--f,v-ratitcboolidvDaiatwoinshhlcynriirpne-igsdiereCtdPtigauPaieeidhvvaa-iscaabsAnhrepaiatlcctstbaiiymghPoGhoteyozloidedemernenniEexirod-ebdcsgnoppylhnaoeozxlnapabiywanefgoynyeotydspxdfstimbtppertesoPlhhcuNto(loeerdioAeFatgdnun-rAiytnopieueasgdGsegrDnhnssmaeon.Eoeluoadtrb+-zy3efsslifdeaydAftepAii(aofsmrensFahedcd,saatmpiorsetteeenginaioo-ltccnav.didpfenddoytploi4hceelirtliruTfAetytdohddsrehmcx)syaeutad(vceh.pohrbdFlgeoeahapfdArelmirnmaneofoegroslttzepgglud.aemIpeeiyateep)e3npsrlim(coen.codBtiFttWxsdayaidiaei6i,tnommtslosge-c0(ileficpenahso.looteeaot,hun4hymmnbirhnmole(tdaBrifehFpiPesalprpan,4ipeloitpD3ritltlegedy)hraogeeo)xid...lnotiengl.y,e(ni3yWbenlaegFipAeatsspeinIihieitsneg)si)intoiped.etna.hodagnitAca3nopiridce(dtcgenBrPocleioeeteavtowmrtfi)lsusenvoafib.bethnapcTtanieanotielolettaonhlhecetn2enayshxneddeesr,),e Pronase, a broadspectrumprotease,resultsin progressive proteolysis with carboxypeptidase A gives rise to a splitting loss of the ability to reactivate the apodehydrogenase with of the original band. The active enzyme-phospholicpoimd plex is contaminantpresentinthe original sample (see Fig. 5B). Inactivatedby Pronase the rateof inactivation With carboxypeptidase A, the enzyme-phospholipid complex is much more slowly inactivated than theapodehydrogenase, Portions of this paper (including “Experimental Procedures,”part the phospholipidaffordingmajor protection (80%) against of “Results,” and Figs. 1-9) are presented in miniprint at theend of loss of activity (Fig. 4A) and proteolysis (Fig. 4B, lane 2 ). Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. D-P-Hydroxybutyrate apodehydrogenase was not inactivatedby three otherexopeptidases, carboxypeptidase B, carboxypeptidase P, andleucine aminopeptidase K, i.e. the enzyme could be fully reactivated. D-&Hydroxybutyrate apodehydrogenase (Apo-BDH)or the enzyme-phospholipid complex (BDH-MPL) was. Digestions were carried out at room temperature (24 1“C)in the absence or presence of 5 mM NAD+as indicated
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