Molecular Weight of Native and Dissociated Cysteamine Oxygenase

Abstract

Cystearnine oxygenase has been further purified to a degree where it appeared homogeneous in disc gel electrophoresis. The purified enzyme possesses a molecular weight of 52000 when it is treated with either 8 M urea or with 0.1 O/, sodium dodecyl sulfate. The molecular weight of the native undissociated enzyme was 96000 when determined by dextran gel filtration and 92000 when determined by sedimentation equilibrium in the ultracentrifuge. These results are consistent with the occurrence of two similar subunits in the molecule of cysteamine oxygenase.I n recent works of this laboratory [1,2] data were obtained suggesting the subunit composition of cysteamine oxygenase. The present paper is aimed at the demonstration of the occurrence of subunits in this enzyme and a t the determination of their molecular weight. Advantage has been taken for this purpose of recent procedures developed for the study of protein subunits, namely, molecular sieve through Sephadex in the presence of 8 M urea [3] and rate of migration on polyacrylamide gel in the presence of sodium dodecyl sulfate [4]. Both the procedures gave comparable results consistent with the occurrence of two subunits in the enzyme. The molecular weight of the dissociated enzyme however did not fit with the molecular weight of the undissociated enzyme obtained earlier [l]. The molecular weight of the native enzyme was therefore redetermined and found higher than that previously reported. MATERIALS AND METHODS ChemicalsBovine serum albumin was obtained from Calbiochem (Los Angeles) ; aldolase, catalase and ribonuclease from Boehringer Mannheim GmbH (Mannheim) ; pepsin from Fluka (Buchs); chymotrypsinogen A from Sigma (St. Louis) ; ovalbumin was prepared according to a reported method [S]; blue dextran 2000 and Sephadex G-I50 (particle size 40 to 120 p ) were from Pharmacia (Upsala) ; ampholine from LKB (Stockholm). Human hemoglobin was a kind gift from Prof. E. Antonini and N-dinitrophenyl alanine from Dr. F. Bossa Dextran Gel FiltrationMolecular weight of the native enzyme was determined on Sephadex G-150 at 4", equilibrated and eluted with 0.01 M phosphate buffer pH 7.6, while the molecular weight of the enzyme treated with urea and mercaptoethanol was analyzed at 20" on Sephadex G-150 eluted with 0.01 M Tris-C1 pH 8.0 containing 8 M urea 0.1 M mercaptoethanol and 0.001 M EDTA, after 12 h of incubation in the same buffer a t room temperature. I n both cases the flow rate was 2.6 ml per hour per cm2, and fractions were collected in sizes of 1 ml per fraction. Blue dextran was used to measure the void volume (V,) and N-dinitrophenylalanine or phenylalanine to measure the inner volume (Vi). I n the case of the column operated in the presence of mercaptoethanol the eluted fractions were assayed by a turbidometric procedure. Each fraction was added with I ml water and 0.2 ml trichloracetic acid to a final 5O/, conceiitration and measured a t 450nm. Proteins in the absence of mercaptoethanol were monitored in the conventional manner. Blue dextran was located by meas...

Mitochondrial Aspartate Aminotransferase from Beef Kidney

“…The molecular weight was determined by a chromatographic procedure, performed following the method of Andrews [18] with the modification of Fish [19] and Cavallini [20] on Sephadex G-150 using as references egg albumin, blue dextran, bovin albumin and cytochrome c. The elution volumes were transformed to the corresponding kd values and these latter were plotted against molecular weight according to Gelotte [21].…”