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The Purification and Properties of a Hexokinase from the Corn Scutellum

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The Purification and Properties of a Hexokinase from the Corn Scutellum THE PURIFICATION AND PROPERTIES OF A HEXOKINASE FROM THE CORN SCUTELLUM By HERBERT CHARLES JONES HI A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTL^L FULFILLMENT OF THE REQUIREMENTS FOR THE DEC»EE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA June, 1965 Dr. T. E. !l!he vriter vlshes to express sincere appreciation to HuEsihreys for his help, guidame, leadership, patience and for the tise of his laboratory facilities in the course of this rssearchj to Dr. G. Ray Hbggle who inspired tte vriter to pursue graduate study; to Drs. D. S. Anthony, R. H. Biggs and T. ¥. Steams for their aid and service on the committee j and to the Departoent of Botany and the Departnent of Health, Education and Welfare for financial support throu^ National Defense Education Act ELtle IV and Hational Institutes of Health feUxswships. JTTp AGRi- CULTURAl LIBPA'-V UNIVERSITY OF FLORIDA TABLE OF COOTEHTS li Lisa? OP TABLES v LIST OP FIGfUR^ vi HJTBCBUCTION 1 BSVim GF LZTSBATORE 3 MATERIALS AHD MEKIODS 36 Plant Materials Prepai^tion of the Enzyme Extraction Annnonimn sulfate fractioiation Absorption and elution froa alumina C-y gel Assajf }§3thods Method 1 Ifethod 2 Assay for phosphofruetokinase, phosphoglucomutsise and glucose -6-phosphatase activities Protein Determination Chemicals and £nz;yines Page RESULTS k2 Purification Substrate Specificity Nucleoside triphosphates Metal Activators Inhiljitozv Sugaars Nucleoside di- and triphosphates Sugar phosphates Anions pH and Temperature Optima DISCUSSION 7^ SUMMARY 80 BIBLIOGRAPHY 8l BIOGRAPHICAL SKETCH 89 ir LIST OF TABIDS 1. MtCHAELIS COSBTASITS {Km) AND RSLATIVS J^JAXIMAL RASES FOR BRAIN AM) YEAST HEXOiCCKASE 7 2. SFFECTS OP GLUC0SE-6-P AMD RELATEID COMPOUNDS OSS PHOSPHOSSLATIOHS BY BRAIN HSXOKDJASE 9 3. PURIPICATIOI OP ffiiXOKIHASE ^3 k, ATPASE ACnVTIY OP THE HEXDKIMSE PREPARATIONS 2^5 5. SUBSTRAOS SPSCIFIC33?3f OiP CORN SCOTELLUM EEKOKENABE 49 6. EFFECT OP !3W;LEX)SIDE35U[PH^mTSS AS SilBSTrRHSS FOR ATP 55 7. ACTIVATION OP HEXOKUmSE BY METAL IONS 60 8. EFFECT OF NUCLEOSIDE DI- AND TRIPHOSPHATBS AS INHBITOaS OP ATP IS THE HEXOKISASE REACnCU 68 UST QPFICRJRSS Figuare Page 1. Effect of glucose concentration on the rate of phosphorylation 52 2. Lii^T?eaver-Burk plot of the effect of glucose concentration on the rate of phosphorylation » ^ 3. Effect of ATP concentration on the rate of phosphorylation, and coJDpetitive inhibition by ADP and AMP 57 i^. Linaveaver-^nrk plots of the effect of ATP concentration on the rate of phosphorylation, and competitive inhibition by ADP and AMP 59 5. Effects of Co"*"*" and Mn++ concentrations on tl^ rate of phosphorylation fe 6. Effect of Mg"*^ concentration on the rate of phosphorylation 62 7. Line'v^aver-Burk plots of the effect of l^"^, Co"^ and Ifa"*^ concentration on the rate of phosphorylation 6h 8. Lineweaver-Burk plots of the competitive inhibition of glucose phosphorylation by xylose and H*- acetylglucosamine 6f 9. Effect of pH on the rate of phosphorylation escpressed as the per ceaat of the aaximuEi rate obtained 71 10. Effect of tamperature on the rate of phosphorylation 73 IHTRODUCnON The properties of plant enzymes, especially those of higher isolated plants, are less veil known than those of similar enzymes from animal tissues. It has been assvuoed that the properties associated vlth the characterized animal and lover plant (yeast) enzymes are the same for higher plants. This may be the case, but in order to it is necessary that the plant enzymes be characterized in gain a better understanding of the similarities and differences metabolism between plants and animals. of The enzyme, hexokinase, which catalyzes the phosphorylation ion, glucose in the presence of adenosine-5' -triphosphate and magnesium in both occupies an important position in the metabolism of sugars evidence plants and animals. Humphreys and Garrard {kl) have presented control- which suggests that the hexokinase reaction may be important in organ ling the rate of glucose uptake by the com scutellumj an seed, positioned between the root-shoot axis and endosperm of the com sucrose which where glucose absorbed from the endosperm is converted to during gennina- is subsequently translocated to the developing seedling -6-phosphate competitively tion (28). Their data indicates that glucose uptake in inhibits an enzymatic step associated with net glucose be the scutelluia slices, and they suggest that the step might been hexokinase-catalyzed phosphorylation of glucose. Since it has extent, demonstrated that brain hexokinase (15, I6) and to a small although yeast hexokinase (33), is inhibited by glucose-6-phosphate, 1 2 noncompetltlvely. It seemed desirable to investigate the properties of com scutellum hexokinase. REVIEW OF LITERATURE Historical In 1927, Msyerhof (73) gave the naias, hexoMnase, to an alcohol precipitable fraction of autolyzed yeast vhich when added to extracts of aged frog or rabbit Dsusele greatly enhanced glycolysis. Independ- ently, in 1935^ Euler and Mler (29) and latwak-i'fejin and Mann (66), Isolated an enzyme from yeast—heterophosphatese—which catalyzed the following reaction in the presence of Mg "*"•": Hexose + ATP >Hexo3e-6-phosphate + ADP tfeyerhof (jk), in a description of the hexokinase reaction the same year, reported that the enzyme descrihed "by the two groups was the sazse one responsible for the activity of his original preparation. Kalckar (51), in 1939* demonstrated that kidney extracts phosphorylated glucose and fructose amd, in 1^, Gelger (36) obsei'ved that extracts from brain tissue phosphorylated fructose, mannose and glucose. Belitzer and Golovskaya (5), the same yeax, showed that muscle tissue contained hexokinase, which in the presence of glucose and creatnine catalyzed the phosphorylation of glucose to l«xose-6« phosphate. In 19*H> Ochoa (78) showed the presence of hexokinase in acetone powder of rat brain. The acetone partially Inactivated adenosinetriphosphatase (ATPase). The hexokinase required l^ and transferred phosphate from A!H> to glucose without the liberation of free oirthoi^iosphate 3 . k Colowick et al. (l2), in 19^1, demonstrated that phosphorylation of glucose preceded the oxidation of glucose in heart muscle and kidney extracts and that hexokinase catalyzed phosphorylation of mannose to niannose-6-phosphate (M6P). The M6P was in turn converted to fructose-6-phosphate (F€P) by an isomerase. In 19'^6, Berger et ^. (6) and Rinitz and MacDonsuLd (57) succeeded in crystallizing hexokinase from, ysast, the only organism frcMa which it has been crystallized. The hexokinases from various sources have been recently reviewed by Crane (20) Hexokinases of Vgarious Animal Tissues Ifammalian brain hexokinases * Colowick et al. (lU) prepared purified beef brain hexokinase by eluting acetone powder fran beef brain with water and f3rau;tional precipitation of the eluate with anaaonium sulfate. The hexokinase was precipitated between 30 and 50 per cent saturation with a two-fold increase in specific activity. The 30 to 50 per cent fraction could be purified further by refractionation between k3 and 50 per cent saturation with annaonium sulfate. The product of the beef brain hexokinase reaction was shown to be glucose -6-phosphate (Gr6P). Jfeyerhof and Wilson (75) observed that brain extracts catalyzed the phosphorylation of glucose and fructose at about the same rate at 0.020M concentrations of the substrates, while at concentrations of 0.0015M to 0.003M the rate was the san» for glucose and much lower for fructose. They observed the same difference relative to the AK» concentiration and there was a greater A2Pase activity in the presence of fructose than glucose. They suggested that there are actually two separate enzymes in brain extracts—a glucokinase and a fructokinase . 5 Wiebelhous and Lardy (icA) noted that dialyzed extracts of "beef brain showed different hexokinase activities with respect to glucose and fructose in the presence of inhibitory concentrations of sodium salts. Sodium salts inhibited phosphorylation of glucose^ but not of fructose, indicating the possibility of two different heMjlsinases. Iheir beef brain extracts promoted the phosphoxylation of glucose, fructose and to a lesser extent, mannose, but were not active with L-glucose, galactose, L-sorbose, D-gluconate, S-keto-O-glucoaate, D-ribose, D-arabinose, L-rhamnose and D-xylose. Inorganic pyrophosphate inhibited activity ^ to 80 per cent depending on its coTOentration. Magnesium ion, up to two times the concentration of the pyrophosphate, did jaot reverse inhibition. Orthophosphate was only slightly inhibitory. Long (63) found that of five rat tissues—brain, liver, kidney, steletal muscle, and intestine—brain had the greatest amount of hexokinase activity, while liver had the least. Crane and Sols (16, 17, 86) prepared particulate he:a>kinase from brain, which was free of interfering enzymes, by fractional centrifuga- tion and solubilization of the enzyme with lipase and/or deoxycholate They obtained 35 per cent recovery. With this preparation, it was found (as V^il-M&Iherbe and Bone (102) had reported in 195X) that g6P inhibited hexose phosphorylation noncompetitively (15), while ADP behaved as a competitive inhibitor (85). They (86) examined the specificity of brain hexokinase toward thirty-five compounds structur- ally related to glucose in order to deteimine which groups of the glucose molecule were possibly involved in formation of a glucose- brain hexokinase complex. Sixteen of the analogs served as substrates . 6 (Tab3je l), five behaved as competitive inhibitors, and the remainder vere inactive. They estimated the relative influence of each hydrojcyl group of glucose by (^mgaxing the Michaelis constants (Kia) and the relative maximal rates (VtaMc) of phosphorylation (Table l). The con- clusion was that the formation of a glucose -enzyme ccxuplex involved the ring structure and hydroxyl giroups at carbon atoms 1, 3, k and 6, and that each hydroxyl group had a specific quantitative influence on enzyme-substrate affinity. Crane and Sols (l6) investigated the specificity for inhibition of the brain hexokinase reaction by G6P and related compounds. They tested some twenty-five compounds and only six were inhibitory (Table 2).
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