Role of Adenosine Triphosphate in the Hemolysis of Sheep Erythrocytes by Typhus Rickettsiae

Hemolysis bytyphus rickettsiae wasfirst reported byClarke andFox(1948), whofound that homogenates ofyolksacsinfected withthese organisms brought about aslowlysis ofsheep and rabbit erythrocytes at37C.Snyder etal.(1954) laterdescribed a faster spectrophotometric methodformeasurement ofhemolysis bytyphus rickettsiae, andshowed that thehemolytic activityofdifferent preparations oftyphus rickettsiae, bothcrude andpurified, paralleled their toxicity formice. Theyalsofoundthathemolysis by typhus rickettsiae required thepresence ofglutamate andMgandwasprevented bysuchmetabolic inhibitors ascyanide andfluoride. Morerecent workhasshownthathemolysis carried outinthepresence ofglutamate, asoriginally described, isinhibited byDNP,2andthat frequently ATP canserve apartial substitute forglutamate. Theamountofhemolysis isloweredwhenglutamate isreplaced byATP,but suchhemolysis asdoes occur isnolonger inhibited bycyanide orDNP.Itwasalso noted thatrickettsiae subjected tocertain procedures thatlead toareversible loss ofrespiratory activity (aswell astoloss oftoxicity andinfectivity) could no longer hemolyze inthepresence ofglutamate, but could dosointhepresence ofATP.These results suggested that ATPmaybeinvolved insomeessential capacity inthehemolytic reaction; fully viable rickettsiae being abletoformtheir own ATPinthepresence ofglutamate, those that had lost theability tooxidize glutamate needing an 1Thisworkwassupported byaresearch grant (E-167C7) fromtheDivision ofResearch Grants, National Institutes ofHealth, U.S.Public Health Service. 2Thefollowing abbreviations areused:ATP, adenosine triphosphate; ADP,adenosine diphosphate; UTP,uridine triphosphate; CTP,cytidine triphosphate; GTP, guanosine triphosphate; DPN, diphosphopyridine nucleotide; CoA,coenzymeA; GSH, glutathione, reducedform; DNP,dinitrophenol. external source ofATP.Additional variations of this phenomenon, that cannot beexplained solely intermsofthis hypothesis, havebeenfoundin theresponse ofdifferent types ofrickettsial preparations toaddedATP. Thehemolytic reaction hastherefore been studied withrespect totheability ofvarious preparations ofrickettsiae toproduce hemolysis inthe presence ofthefollowing substrates: (1)glutamate,whichcanserve asasource ofinternally generated ATP;(2)externally addedATP,usually withKCN added also toprevent generation ofATP;and(3)glutamate andATPtogether. Withregard totherelative amountofhemolysis produced underthese three conditions, therickettsiae fell intofourcategories: (A)undamaged,