Worthington: characteristics and other parameters of hexokinase from yeast

AI Meijie Worthington Hexokinase enzymatic reaction ：

 

Hexokinase is isolated from yeast cells in two different forms , be called  PI  and  P-II (Schulze et al . 1969). These are independent 、 Isozymes that cannot be converted to each other （Womack wait forsomeone 1973）.

Worthington  Characteristics of hexokinase from yeast ：

Molecular weight ： The molecular weight of the natural form is about  100,000 (Schulze et al . 1969), The molecular weight is slightly higher than  50,000 (Schmidt et al . 1973)  The polypeptide chain of .

The best  pH  value ：7.5 - 9.0 (Sols et al . 1958).

form ： PI  and  P-II  Both contain the same amino terminal valine and the same carboxyl terminal alanine .Schmidt Amino acid composition has been reported by et al .（1973b）.

Extinction coefficient ： Extinction coefficient PI  by  8.85,P-II  by  9.47（Schmidt wait forsomeone 1973）.

Isoelectric point ：PI, 5.25  and  P-II, 4.93 (Schmidt et al . 1973).

Inhibitors ： Enzyme coated with  SH  Compounds that inhibit group reactions . It was also  1- Sorbose phosphate 、 Polyphosphate 、6- deoxidation -6- Fluoroglucose 、2-C- hydroxyl - Methyl glucose 、 Xylose and lysozyme inhibit （Sols  wait forsomeone  1958  and  McDonald 1955）.

Activator ： The catalytic activity of hexokinase requires magnesium ion . It is activated by catecholamines and related compounds  (Harrison et al . 1972). Calcium ion does not affect enzyme activity .

Specificity ： The enzyme phosphorylates  D- Fructose 、5- Keto -D- Fructose （Avigrad wait forsomeone 1968）、D- glucose 、2- deoxidation -D- glucose 、D- Mannose and  D- Glucosamine . It has been proved that  ATP  and  ITP  Transphosphorylation occurs in yeast hexokinase reaction  (Martinez 1961).PI  The activity with fructose is glucose  2.6  times , and  P-II  Fructose : The glucose ratio is  1:3 (Lazarus et al . 1966).Bessell The substrate specificity of yeast hexokinase has been extensively studied by et al .（1972）.

stability ： Freeze dried preparations and crystalline suspensions are in  2-8°C  Stable under  6-12  Months .

AI Meijie Worthington Hexokinase assay ：

Method :  The determination is based on passing with glucose -6- Coupling reaction of phosphate dehydrogenase Reduce NAD + , And by measuring 340 nm The increase of absorbance at... Was determined by spectrophotometry .

Under specified conditions , stay  30°C  and  pH 8.0  Under the condition of , One active unit can be reduced per minute  1  Micromore  NAD + .

Reagents ：

1.0.05 M Tris*HCl  Buffer ,pH 8.0, contain  13.3 mM MgCl 2

2.0.67 M  Glucose is above  Tris⋅MgCl 2 In buffer

3.16.5 mM  adenosine  5' Triphosphate is in the above  Tris⋅MgCl 2 In buffer

4.6.8 mM NAD  In the above  Tris⋅MgCl 2 In buffer

Be careful ：NAD  The salt form and degree of hydration may vary . Analytical grade and correct molecular weight should be used with care .

Leuconostoc mesenterica glucose -6- Phosphate dehydrogenase （Worthington  Code ：ZF  or  ZFL）. With  300 IU/ml  The concentration of dissolved in the above  Tris⋅MgCl 2 In buffer . Store in  0 - 4°C.

enzyme ：

Dissolve in  Tris⋅MgCl 2 In buffer ,pH 8.0  In order to obtain  0.02 - 0.04 ΔA/min  Rate .

Program ：

Adjust the spectrophotometer to  340 nm  and  30°C.

Move the pipette into each cuvette , As shown below ：

Tris⋅MgCl2  Buffer  2.28  ml

0.67 M  glucose  0.50  ml

16.5  Mm adenosine triphosphate  0.10  ml

6.8  mm  NAD 0.10  ml

G-6-PDH 0.01  ml

In the spectrophotometer  30°C  Incubate  6-8  Minutes to reach temperature equilibrium and establish blank rate （ If there is ）. At zero o'clock , Join in  0.1 ml  Dilute hexokinase solution and mix thoroughly . Record  A 340 increase 3-4  minute . Determine from the initial linear part of the curve ΔA/min.