Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 1960 Calcium Effects on Yeast Hexokinase Mary Ann Hurley Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Part of the Medicine and Health Sciences Commons Recommended Citation Hurley, Mary Ann, "Calcium Effects on Yeast Hexokinase" (1960). Master's Theses. 1522. https://ecommons.luc.edu/luc_theses/1522 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1960 Mary Ann Hurley CALC IUM EFFECTS ON YEAST HEXOKINASE by Mary Ann Hurley ' .... "-' '., ! A. Thesis Submitted to the Faculty of the Graduate School of Loyola University in Partial Fulfillment of the Requirements tor the Degree of }Bater of Bcience February 1960 LIFE Mary Ann Hurley 'eTaS born in Cleveland, Ohio, February 4" 1934. She was graduated from Woodrow Wilson High School, Washington, D. C., June .. 1951 and from Trinity College .. Washington, D. C., June, 1955, with the degree ot Bachelor of Arts. ., From July.. 1955 to August, 195".. the author was employed as chemist at the National Heart Institute, National Institutes of Health, Bethesda .. Maryland. She began graduate studies at wyola University in September, 1957 .. holding the position of teaching assistant in the Department ot Biochemistry for the academic years, 1957-1958 and 1958-1959. ii The author wisbe. to thank Dr. Norten C. .Melchior tor hi. continual guidance and kindnes •• 1ii TABLE OF CONTENTS Chapter I. IRTRODUCTiON ••••••••••••••••••••••••••••••••••••••••••••• 1 Properties ot yeast hexokil'l&se -- ATP c~lexe. -­ Theories ot the mode ot action ot ~TP- ..• Inhibition b7 calc ium. II. • ••••••••••••••••••••••••••••••••••• 9 Preparation ot solutions -- Isolation and puritication ot yeast hexokinase -- Evaluation ot the enzyme pre­ paration -- Method ot calculation -- Protein detel'll1n­ ation -- Speetrophoto.etric meuurementa -- Cell corrections -- Path leDgtha -- Preparation ot ATP -­ Enz~ asaay -- Kinetic Studies _. pH measurements -­ Preparation ot ion exchange re.in -- U.e ot Et41Br. III. EXPLORAmRY ENZYME STUDIES • •••••••••••••••••••••••••••••• IV. RESU~ •••••••••••••••••••••••••••••••••••••••••••••••••• 40 Eftects ot varying the concentration ot -cneaium &J.:1d calcium on the yeast hexokinase s;yatea. v. DISCUSSION • •••••••••••••••••••••••••••••••••••••••••••••• VI. SUNMARY •••••••••••••••••••••••••••••••••••••••••••••••••• 53 BIBLIOGRAPHY •••••••••••••••••••••••••••••••••••••••••••••••••••••• 54 APPElmIX I •••••••••••••••••••••••••••••••••••••••••••••••••••••••• 57 iv LIST OF TABLES Table I. COMPOSITION OF BUFFER-DYE SOWTIONS FOR KINETIC S'lUDIES •• 11 II. RELATIVE ACTIVITIES OBTAINED DURING PREPARATION OF HIGH SPECIFIC ACTIVITY HEXOKINASE ••••••••••••••••••••••••••••• 16 III. SPECTROPHOTOMEmIC TITRATION OF BUFFER-DYE SOLU'l'IOBS ••••• 19 IV. DEPENDENCE OF ACID PRODUCTION UPON GWCOSE ••••••••••••••• 26 V. ACID PRODUCTION IN ABSENCE OF ENZYME ••••••••••••••••••••• 36 VI. THE EFFECT OF ALBUMIN ON THE REACTION RATE OF HEXOKINASE. 31 VII. THE EFFECT OF AIBUMIN ON THE pK OF CRESOL RED •••••••••••• 38 " VIII. THE EFFECT OF ALBtOO:N ON THE ABSORPTION PEAK OF CRmOL RED 39 IX. THE EFFECT OF MAGNESIUM ON THE VELOCITY AND IONIC SPECIES WITH 2 aM ATP •••••••••••••••••••••••••••••••••••••••••••• 41 X. THE EFFECT OF MAGNmIUM ON THE VELOCITY AND IONIC SPECIES WITH 5 aM ATP ••••••••••••••••••••••••••••••••••••••••••• 42 XI. THE EFFECT OF CAlCIUM ON TBE VELOCITY AND IONIC SPECIES WITH 2 aM ATP •••••••••••••••••••••••••••••••••••••••••••• 43 XII. THE EFFECT OF CAlCIUM ON THE VELOCITY AND IONIC SPECIES WITH 5 aM ATP •••••••••••••••••••••••••••••••••••••••••••• 44 XIII. THE EFFECT OF MAGNESIUM ON ACID PRODUCTION ••••••••••••••• 51 XIV. THE EFFECT OF CAICIUM ON ACID PRODUCTION" I ••••••••••••••• 58 XV. THE EFFECT OF CAWIUM ON ACID PRODUCTION II •••••••••••••• 59 XVI. THE EFFECT OF CAlCIUM ON ACID PRODUCTION III ••••••••••••• 62 v LIST OF FIGURES Figure Page 1 THE EFFECT OF CALCIUM CHLORIDE ON IONS IN ATP SOWTIONS ti 2 TEST OF OPTICAL :MEASUREMENT OF' ;ACID ••••••••••••••••••• 21 3 EFFECT OF ENZYME CONCENTRATION ON ACID PRODUCTION ••••• 24 4 DEPENDENCE OF ACID PRODUCTION UPON GWCOSE •••••••••••• 25 5 THE EFFECT OF MAGNESIUM ON ACID PRODUCTION •••••••••••• 27 6 EFFECT OF CAtcIUM CHLORIDE ON ACID PRODUCTION BY llEltOKI.NA8E ••••••••' •••••••••••••••••••••••••••••••••••• 45 7 THE EFFECT OF CALCIUM ON HEXOKINASE ACTIVITY: •••••••••• 51 8 TJIE EFFECT OF CALCIUM ON HEXOKINASE ACTIVITY: •••••••••• 52 vi CHAPTER I INTRODUCTION An absolute requirement in 11ving matter is some mechanism whereby the tree energy liberated in the oxidation ot organic metabolites is trapped so that it can be transferred and used tor the various vital processes which require energy. The work ot Meyerhot ('27, '34), Lundsgaard ('30a, '30b, '31), Lohmann ('31, '34), Warburg ('39,'41) and the Coris ('40), served as the toundat1o~tor uncovering such a mechanism which depends on the formation of energy-rich phosphate compounds. It became apparent trOID their work and the work ot succeeding investigators that adenosiIlf:triphosphate (ATP) is a uniquely important molecule. It is essential for the biosynthesis of organic molecules, mechanical work and work against osmotic rorcee in absorption and secretion. As part ot extensive research into the nature ot high-energy phosphates many investigators bave studied the reaction catalyzed by hexokinase as defined by Colowiek and Kalckar in 1943: O· O· 0- R .. OR + R' .. 0 .. p+. 0 - p+.. 0 .. p+- 0- ) O· 0- 0- Glucose ATP o- 0- o· R .. 0 - P+- 0- + R' .. 0 - p+- 0 .. p+- o· + O· 0- o· Glucose .. 6 .. phOsphate ADP 1 2 The enz~ which catalyzes this reaction in feast bas 'been obtained in crystalline :torm by several. workers.. Yeaat hexokinase is a protein with a molecular weight ot 96,600, an isoelectric point at pH 4.5 - 4.8, and con­ tains three atoms of phosphorus per mole. (Kunitz and kDonald, '46). In the presence ot ATP and -anesiUll, the enzyae phosphorylates slucose, tructose and mannose. The attinity for glucose is much greater than that tor fructose, (xm::; 1 X 10-4 and 7 X 10-4 moles per liter, respectively) but V-.x with tructose 1s tw1ce that with glucose. The enzyme is stable 1n the pH range frO'll 5 to 8, With activit,- rapidly lost at pH'a above 9 or below 4. According to Sols et al ('58), the enzyme po.sesses highest activit,- at pH " 7.5 with about one-halt axial activity at pH 5.4 and 9.4. Darrow ('57), however, statel that the activity at pH 8.5 il at least two tu.s the activit,- at pH 7.5. The equil1briWi conataat ot the reaction as determined b,- Robbins and Boyer ('57) il 3.86 X 102 at pH 6.0 and increases by a factor of approx1-.tely 10 per pH unit in the region of pH 6 to 8. Darrow has recently developed a more convenient procedure tor obtain- ing a crystalline preparation which is reported to be tree trom contaDdnat-­ ina pyrophosphatase, phosphobezose 1soaerase and triosephosphate dehydrogen­ ase which contaminated previous preparations. (Robbins and Boyer, •57) • The enzyme requires aglle.1um tor act1vity (See Figure 5). This re- qu1rement has usually been presented as due to the formation of a magnesium- enzyme complex and the appropriate Michaelis conatant baa been evaluated by exper1llents in wh1ch all other substances (except the enzyme) were present 1n excess. --- 3 The demonstration that ATP forme complexes with _~ positive ions in- cluding magnesium torces a re-evaluation ot the assUDlption that the agtlesium tunctions o:11y as an enzyme activator. D1.Stetano and leumann ('53) studied the d1str1but1on of calc1um 45 be­ tween 10n exchange res1n aDd aqueou solut10n w1th and w1thout ATP. They tound that the add1tion ot ATP to a tiXed amount of cat10n and reain 1n­ creased the amount ot cat10n which vas removed f'rom the res1n, 1ndicat1ng a calc1um-nucleot1de coaple.x. Their data ind1cated that a mooo-_tal-DlOno­ nucleot1de cOJlple.x .s the D8j:)r f'orm ot the comple.x, at lealt in dUute solutioD8. Sp1cer ('52) wh11e studying the ef'fects of' pH, 10nic strength on the contractile phases of the acto~sin response to ATP found that add1tion ot calc1um or agnes1U11 ions to the sol.utions ot ac~sin containing ATP resulted in a decrease in pH. Be also noted that in the presence of' either or both of' these ions, there is an increase in the rate ot hydrolya1s of 0 ATP at 100 • Burton and Krebs ('53) est1Jlated the formation constant ot MgATP",2 comple.x by the titration technique. I. C. Melchior ('54) by titrating tetraalltyl..uaon1um ATP with aDd without sodium or potasSium ions found that in certain pH ranges, less hydrochloric acid was required to reach a given pH in the presence of these ions than in thei,).· absence, indicating comple.x formation. Martell aDd SchYarzenbach ('56) alao by the titration _thod showed that ATP as well aa other polyphospbatea formed comple.xea with calcium and _gnesiUll ions. --- Smith and Alberty ('56) deaonstrated coaplex toration ot ATP nth lithium, sodiU1ll and potusiUII by the titration technique, and Wal.aas ('58) by the ion-exchange method foUDd that calcium and magnesiUJI fo1'll IIOno ...metal­ mono-nucleot1de complexes With ATP and other nucleotides. Several authors bave attempted