Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.
Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.
Mocali
A
A
Istituto di Patologia Generale, Firenze, Italy.
Paoletti
F
F
D000937:Antigen-Antibody Reactions; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D005779:Immunodiffusion; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007962:Leukocytes / Q000201:enzymology*; D008214:Lymphocytes / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology; D014174:Transketolase / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*
D007106:Immune Sera; D014174:Transketolase