Biomedical Research 2 (1) 1-3, 1981
i RATES OF DISSOCIATION OF PYRIDOXAL 5’-PHOSPHATE FROM GLUTAMIC OXALOACETIC TRANSAMINASE IN A NORMAL AND DEFICIENT STATE
SATOSHI SHIZUKUISHI, KINZO TAKEMURA and KARL FOLKERS* Institute for Biomedical Research, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
ABSTRACT A study has been made of the relative rates of dissociation of pyridoxal 5’-phosphate (the coenzyme) from the glutamic oxaloacetic transaminase of erythrocytes (EGOT) from the blood samples of subjects who had low basal specific activities and high deficiencies of activity, and from blood samples of subjects showing normal specific activities and no deficiency. The rates of dissociation of the coenzyme from a state of deficiency of vitamin B6 was l.6i0.9 and the corresponding value for a state of no deficiency was 1.4 1: 0.4 (n.s.). These data support the interpretation that the increase in the specific activity of EGOT by oral pyridoxine may result from an increase in the synthesis of the apoenzyme by transcriptional and translational mechanisms rather than by decrease in the rate of degradation of the apoenzyme.
KEY WORDS vitamin B6 / pyridoxal 5’-phosphate/ GOT / deficiency / dissociation
In a series of studies (2, 3, 6, 7, 10) on a human S.A. to ca. 0.7. deficiency of vitamin B6, the following apparent Oral pyridoxine does not function like a facts have emerged: (a) The existence of a drug with a pharmacodynamic action, but deficiency of vitamin B6 in the population is rather is converted to pyridoxal 5’-phosphate very common and may even approach univer- which functions as a coenzyme. The continuous sality in the absence of oral supplementation by increase in the enzyme activity of EGOT from pyridoxine; to paraphrase, the diet is generally a deficiency state to a normal state is associated inadequate to provide an adequate intake of with clinical improvement of the carpal tunnel vitamin B6 for the best health. (b) The carpal syndrome, and requires weeks of therapy. This tunnel syndrome, a neurological compression long-term increase in enzyme activity can result disease, exists widely in the population. (c) either from transcriptional and translational The glutamic oxaloacetic transaminase of the mechanisms yielding more enzyme molecules erythrocytes (EGOT) is an excellent B6-enzyme (11) or from a decrease in the rate of degrada- to measure a state of B6-deficiency or B6-normal- tion of the apoenzyme. cy. (d) Treatment of a subject with a low specif- Litwack and Rosenfield (8) studied enzymatic ic activity (S.A.) of EGOT (i. e., a state of activities as a measure of relative rates of coen- deficiency) invariably leads to an increase in zyme dissociation. Their studies included ala- S.A. over periods up to 12 weeks. (e) A defini- nine aminotransferase and aspartate amino- tive (from a double blind study) clinical im- transferase; both enzymes have pyridoxal 5’- provement of the carpal tunnel syndrome is phosphate as a coenzyme. They utilized activat- invariably associated with an increase in the ed charcoal to remove dissociated pyridoxal 5’-phosphate. Their data showed that the rate of dissociation of the coenzyme was related to *To whom reprint requests should be made the half-life of the enzyme, and indicated that 2 s. SHIZUKUISHI, K. TAKEMURA and K. FOLKERS
dissociation of the coenzyme may be a limiting of distilled water equal to that of the charcoal step in the degradation. suspension (instead of the charcoal suspension) We have investigated the relative rates of was added to the incubation mixture. The dissociation of pyridoxal 5’-phosphate from the filtrate was assayed for the enzymatic activity of EGOT of blood samples from subjects having EGOT, as described (6, 7). a deficiency of vitamin B6 as well as no deficien- The specific activity (S.A.) of the enzyme was cy. expressed as nmoles of pyruvate which was formed/hr/108 erythrocytes. The % association was calculated as follows: % association: [S.A. METHODS without PLP] / [S.A. with PLP] >< 100. The About 12ml of blood was collected from the values of % association were plotted against cubital vein of the subjects into a vacutainer time. The rate of dissociation was calculated containing EDTA. The vacutainers were gently from the slope of the plots by regression rocked to promptly and completely dissolve the analysis. The state of dissociation was expressed anticoagulant. The blood was then centrifuged as — 21% association per hour. for 10 min at 1,500 rpm. The upper transparent layer of plasma and the intermediate layer of leucocytes were discarded. The fraction of RESULTS AND DISCUSSION erythrocytes was suspended in about 8 ml of a The data in Table 1 show that the mean basal solution of 0.9% NaCl. This suspension was S.A. of the glutamic oxaloacetic transminase of centrifuged for 5min at 2,000 rpm, and the the erythrocytes (EGOT) was 0.30d:0.04 for a upper layer was discarded. Washing with group (n:7) of subjects who were not taking about 8ml of the NaCl solution was twice an oral supplement of pyridoxine. The corres- repeated. After centrifugation, the layer of ponding mean value of the basal S.A. was erythrocytes was treated with about 6ml of 0.65i0.07 for a group of subjects (n=7) who 0.9% NaCl solution which was two times the were taking an oral supplement of pyridoxine. volume of the erythrocytes. The number of The difference in the mean values of S.A. is erythrocytes in the suspension was counted by significant, P<0.001. Similarly, the difference a Coulter Counter (Coulter Electonics, Inc., between the mean values of % deficiency of Hialeah, FL). The suspension of erythrocytes activity for these two groups is significant, was hemolyzed by diluting with 56 ml of 0.01 M P<0.001; Table 1. potassium phosphate buffer, pH 7.4, which was The rate of dissociation of pyridoxal 5’- seven times the volume of the suspension of phosphate over 4hr of time, as measured at erythrocytes. 0, 1, 3 and 4-hr intervals, was measured for the The rate of dissociation of pyridoxal 5’-phos- EGOT from blood samples of the subjects in phate from EGOT in the absence and in the the two groups. The dissociation rate was presence of activated charcoal (Fisher Scientific 1.6i0.9 (— 21% association/hr) for the subjects Co., Fair Lawn, NJ) was measured according having a deficiency of vitamin B6, and the rate to Litwack and Rosenfield (8). A suspension of was 1.4:t0.4 for the subjects having no deficien- 100 pl of charcoal in distilled water at a concen- cy; the difference in these rates is not significant. tration of 100 mg/ml was added per ml of the These data are interpreted on the basis that hemolysate. Fifty-two ml of the hemolysate the increase in the values of S.A. of EGOT when had been “treated with 5.2 ml of charcoal sus- subjects are treated with oral pyridoxine results pension to remove endogenous-free pyridoxal from increases in the synthesis of the apoenzyme 5’-phosphate before initiation of the incuba- probably by transcriptional and translational tion. During incubation, 10ml aliquots were taken at 0, 1, and 3 or 4 hr after initiation of the incubation, and the aliquots were treated with Table 1 lifiect of Oral Pyr:'d0xr'ne Trearmem‘ on EGOT Acrz'vz'iies lml of the charcoal suspension. The mixture containing the charcoal was agitated by a Vortex Deficient state Pyridoxine treated P Mixer (Scientific Products, McGaw Park, IL) (I1 = 7) (I1 = 7) for 20 sec, and it was then centrifuged for 600 g for 2min. The supernatant was centrifuged Basal S.A. 0.30i0.04 0.65 ;t0.07 <0.001 at 12,000g for 5 min, and it was then filtered % Deficiency 35 i 7 8 :|; 4 <0.001 through ca. 400 mg of glass wool in a Pasteur Dissociation rate* 1.6 :l;0.9 1.4:i:0.4 n.s. pipette, 5 mm >< 6 cm. For a control, a volume * -— A% association/hr PYRIDOXAL 5'-PHOSPHATE 3
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