A high specific acitvity form of mammalian liver aldolase

Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28 mumol/min per mg of protein and a fructose 1,6-bisphosphate cleavage of 20-28mumol/min per mg of protein and a frutose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.

Section: Fractionsupporting

confidence: 51%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Purification and properties of the native form of rabbit liver aldolase. Evidence for proteolytic modification after tissue extraction

Chappel¹,

Hoogenraad²,

Holmes³

1978

“…0.2mm or 8mg/ml. It is preferable to remove the substrate from the enzyme as soon as possible after elution to avoid inactivation; this is even more important if aldolase B, the liver enzyme, is isolated by the same procedure (Chappel et al, 1976). Aldolase is also a 'sticky' enzyme, and some salt must be added to the pH8 buffer to loosen its adsorption before adding the substrate.…”

Section: Behaviour Ofindividual Enzymesmentioning

confidence: 99%

Purification of glycolytic enzymes by using affinity-elution chromatography

Scopes¹

1977

101

1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures.

“…In this laboratory all glycolytic enzymes are required in large quantities both for assaying purposes and for metabolic reconstitution studies (Scopes, 1973). Use of the affinity-elution procedure has been widely adopted by us (Scopes & Fifis, 1975;Stewart & Scopes, 1975;Chappel et al, 1976;Scopes, 1977), and the present paper describes procedures for purifying several different enzymes simultaneously from extracts of rabbit muscle.…”

mentioning

confidence: 99%

Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures

Scopes¹

1977

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.