Worthington mammalian lactate dehydrogenase study -- Characteristics and determination scheme

Mammalian lactate dehydrogenase  (LDH)  It exists as five tetramer isoenzymes , It consists of a combination of two different subunits .H Subunits are dominant in myocardium , Suitable for aerobic oxidation of pyruvic acid .M  Subunits are dominant in skeletal muscle , Pay more attention to anaerobic metabolism and pyruvate reduction .

Unit definition ： A unit in  25°C、pH 7.3  Oxidize every minute  1  Micromore  NADH

Please note that , Mammalian lactate dehydrogenase   The source of has changed from natural rabbit muscle form to recombinant expressed in E. coli . The recombinant enzyme meets or exceeds the specifications of the previously provided natural enzyme .

AI Meijie Worthington Ox heart LDH Characteristics ：

Molecular weight ：35,000/ Subunits （Fosmire  and  Timasheff 1972）.136,700 ± 2,100/ Tetramer  (Huston et al . 1972).

form ：Vallee  and  Williams (1975)  It is reported that it is low  pH  Subunit dissociation under . See also  Yang  and  Schwert (1972)  as well as  Gold  and  Segal (1965).

Extinction coefficient ： Extinction coefficient =14.9.

Activities ： See  Borgmann wait forsomeone .（1974）.

Specificity ： This enzyme is effective for  L(+) Lactic acid is specific .Meister (1950)  The report says it can reduce several α- Ketoacids and α,β- Diketone Acid , But the rate of reducing pyruvate is about one tenth .

Inhibitors ：LDH  Quite stable . It is inactivated by iodide . The inhibition of mercury benzoate is very slow . see  Schwert  and  Winer (1963); And Anderson and others .(1974)  and  Bloxham wait forsomeone .（1975  year ）.

Activator ：George Many organic compounds that stabilize enzymes have been reported by et al , For example, dimethyl sulfoxide 、 Ethanol and methanol .（1969  year ）. Diethylstilbestrol and its derivatives can also stabilize enzymes （Cohen et al . 1969）.

From rabbit muscle  LDH  Characteristics of ：

Molecular weight ：140,000.

form ：Lovell  and  Winzor (1974)  The report says , Tetramer in  pH  by  5  Acetate of  -  Completely dissociate into two dimers in chloride buffer （ Molecular weight  70,000）（ Right bull heart  LDH  Conditions without influence ）. Phosphate and pyridine nucleotides stabilize the quaternary structure of the tetramer . Phosphate has activation . See also  Cho  and  Swainsgood (1973).

activity ：Stambaugh  and  Post (1966)  as well as  Zewe  and  Fromm (1965)  The reaction kinetics is reported .

Worthington  Determination of lactate dehydrogenase ：

Method :  The reaction speed is determined by  NADH  Caused by oxidation  340 nm  Determine by reducing the absorbance at . Under specified conditions , stay  25°C  and  pH 7.3  Under the condition of , A unit oxidizes every minute  1  Micromore  NADH.

Reagents :

0.2 M Tris⋅HCl,pH 7.3

6.6 mM NADH  Above  0.2 M Tris⋅HCl  Buffer ,pH 7.3

30 mM  Sodium pyruvate in  0.2  above  Tris⋅HCl  In buffer ,pH 7.3

enzyme :

With  1 mg/ml  Dissolve in  0.2 M Tris⋅HCl  In buffer . Dilute the enzyme before use to obtain  0.02-0.04 ΔA/min  Rate . stay  Tris  In buffer and keep low temperature .

Worthington Determination of lactate dehydrogenase protein concentration in mammals ：

 

Program ：

Set the spectrophotometer to  340 nm  and  25°C.

The pipette is as follows ：

Tris⋅HCl, 0.2 M pH 7.3 2.8  ml

6.6  mm  NADH 0.1  ml

30 mM  Sodium pyruvate  0.1  ml

Incubate in a spectrophotometer  4-5  Minutes to reach temperature equilibrium and establish blank rate （ If there is ）.

Join in  0.1 ml  Properly diluted enzyme , And record from the initial linear part ΔA 340 /min.