Further Studies on Mitochondrial Propionyl Carboxylase

M Daniel Lane,Donald R Halenz,David P Kosow,Carman S Hegre

doi:10.1016/s0021-9258(20)81313-1

Abstract

SUMMARY Propionyl carboxylase from bovine liver mitochondria has been further purified by the use of diethylaminoethyl (DEAE)- cellulose ion exchange chromatography. 2 Report by Dr. S. Ochoa at the Symposium on Metabolic Re- actions Involving Biotin, 137th meeting of the American Chemical Society, Cleveland, 1960. Methybnalonyl-CoA-3-W decarboxylated’ m~moles 0 0 1 2 27 28 8 8 19 17 %t 0 4 83 24 55 * Following incubation, acidified aliquots of the reaction mix- ture were plated and counted acaording to the procedure used for the Cl402 fixation assay (4). The residual radioactivity was con- verted to mpmoles by dividing by the specific activity of methyl- malonyl-CoA-3-W. t Average of duplicates. TABLE VI Effect of arsenate on carboxylation oj propionyl-CoA The reaction mixture contained (in rmoles) : Tris, pH 8.5, 100; propionyl-CoA, 1; ATP and MgC12, 4; GSH, 5; KHW03, 15; and ammonium sulfate-purified carboxylase, 0.05 mg. NazHAs04 added as indicated. Final volume, 1.5 ml. Incubated 20 min- utes at 37”.

Highlights

Coenzyme A, ATP, and ADP were obtained from Pabst Laboratories; avidin, specific activity of 2.5 units per mg (14) and yeast hexokinase, specific activity of 28 units per mg (15) from Nutritional Biochemicals Corporation; yeast hexokinase Type III, specific activity of 160 units per mg (15) and reduced glutathione from Sigma Chemical Company; and DEAE-cellulose from Eastman Organic Chemi
The present investigation does not support either of the mechanisms proposed earlier (4, 7, 10) for the events leading to the formation of the “enzyme-CO;’ intermediate (7) : Enzyme + ATP ti enzyme-ADP + Pi
It should be pointed out that Lynen very recently reported[1] that the exchange of P32-inorganic phosphate into ATP catalyzed by fl-methylcrotonyl carboxylase occurs only in the presence of ADP

Summary

Materials and Methods

Acetyl-, propionyl-, butyryl-, crotonyl-, and valeryl-CoA were prepared from the appropriate anhydride according to the method of Simon and Shemin (11) and methylmalonyl- and P-hydroxybutyryl-CoA according to the method of Wieland and Rueff (12). In the absence of methylmalonyl-CoA or carboxylase, no incorporation of HC140a- occurred It was further demonstrated, by isolation of the reaction products and paper chromatography with methods described earlier (4), that all of the radioactivity was in the form of methylmalonic acid. U4-bicarbonate incorporation into methylmalonyl-Coil in the presence of arsenate and ADP (Table IV, Experiment 3) is probably a result of the formation of propionyl-CoA by reversal of the carboxylation reaction, the formation of ATP from ADP and traces of inorganic phosphate present in certain of the reagents used, and the recarboxylation of propionyl-CoA. Decarboxylation of Methylmalonyl-CoA-To verify the explanation that HCiQ- incorporation into methylmalonyl-CoA occurred through a reversal of the over-all carboxylation reaction, direct evidence for methylmalonyl-CoA decarboxylation was Mitochondrial Propionyl Carboxylase

NYOa- fixed per hour per nog of protein’

Findings

DISCUSSION

SUMMARY