Abstract
Rabbit red blood cell hexokinase (EC 2.7.1.1.) has been purified 300,000-fold by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis. The hexokinase activity has been isolated in 35% yield as a protein that is homogeneous by polyacrylamide and sodium dodecyl sulfate gel electrophoresis. The highest specific activity obtained was 145 units/mg of proteins. The native protein has a molecular weight of 110,000 by gel filtration on Ultrogel AcA 44 and 112,000 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 110,000 indicating that hexokinase is a monomer. The enzyme had a pI of 6.20 to 6.30 pH units by isoelectric focusing. The enzyme was specific for Mg . ATP and Mg . ITP as the nucleotide substrates. Several hexokinase with different affinities.
Highlights
Rabbitredbloodcellhexokinase (EC 2.7.1.1.) has EXPERIMENTALPROCEDURES been purified 300,000-fold by a combination of ionex- Materials-Coenzymes, enzymes, substrates, phenazine methosulchange chromatography, affinity chromatography, andfate, and MTT'were obtained from Sigma
Spectrophotometrically in a system coupled with glucose-6-phosphate dehydrogenase (EC 1.1.1.49)and 6-phosphogluconate dehydrogenase sedimentation velocity on sucrose density gradients
The column was equilibrated with 5 mM sodium potassium phosphate buffer, pH 7.5, containing 3 mM 2mercaptoethanol, 3 mM KF, 5 mM glucose, 0.5 M NaC1, and 9% (v/v) stage the enzyme can be stored at -20°C over a period of weeks, with no significant lossof activity
Summary
The hexokinase activity was located by incubating polyacrylamide gels for 15 to 30 min in a freshly prepared solution containing 0.135 M glycylglycine (pH 8.1), 10 mM glucose, 5 mM ATP. The column was equilibrated with 5 mM sodium potassium phosphate buffer, pH 7.5, containing 3 mM 2mercaptoethanol, 3 mM KF, 5 mM glucose, 0.5 M NaC1, and 9% (v/v) stage the enzyme can be stored at -20°C over a period of weeks, with no significant lossof activity. Purified hexokinase (100pl containing 5 units/ml, specific activity of 145), 10plof aldolase (EC 4.1.2.13) (rabbit muscle, in (NH&S04), 10 I.1 of lactate dehydrogenase (rabbit muscle, in to a Sepharose-N-aminohexanoygllucosamine column (1X 4 cm) equilibrated with the same buffer.After the sample application, the affinity column was washedwith buffer phosphate until the protein absorbance at 280 nm, in the eluate, was lowerthan 0.1 A.The hexokinase was eluted by adding 5 mM glucose to the equilibrating buffer. Cooling temperature was4"C, elution sliced into 0.2-cm sections and separately mashed in 1 ml of flow rate 50 ml/h, and fractions of 1ml were collected and measured for the hexokinase activity and the pH value
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