The enzyme splitting pantethine into pantothenic acid and cystamine has been purified about 3000 times starting from horse kidney. The purification procedure is reported. The enzyme, whose optimal activity lies in the pH range 4.0-5.5, requires the presence of a reduced thiol for the full activity : mercaptoethanol and dithiothreitol give the higher effect. This activation seems not to be specific. The enzyme has a K , value for the substrate of the order of 5 mM and shows inhibition by excess substrate and by products. Substrate specificity studies show that the enzyme splits only the intact substrate molecule, which is not hydrolyzed by several hydrolytic enzymes, even with a broad speci6city (as bacterial pronase and subtilisin). The enzyme could be responsible for the production in vivo of cysteamine, which is known to be oxydized by a specific oxygenase, isolated recently in this laboratory.Identification of an enzyme able of hydrolyzing pantethine t o pantothenic acid and cystaminel has been reported in previous communications from this laboratory [1,2]. The purification of this enzyme is described in this paper. Some characteristic features of the protein and the catalyzed reaction are also presented. The enzyme is named throughout this paper, for our convenience, pantethinase. EXPERIMENTAL PROCEDURE Determination of ProteinThe protein concentration of the starting homogenate was determined by dry weight of samples kept at 110" to constant weight. I n the other steps of purification proteins were determined by the biuret reaction according t o the method of Goa [3].

The substrate specificity of pantethinase, the mammalian enzyme which has been isolated from horse kidney cortex, has been studied. The real substrate has been found to be pantetheine in the reduced form, and the involvement of one or more -SH groups of the enzyme is also considered.A number of other compounds, chemically related t o pantetheine, has also been tested as substrates. Pantethinase is able to metabolise pantetheine-4'-phosphate, with an activity about loo/, of that exhibited for pantetheine ; it is inactive on the other substances tested (pantothenoylcysteine-4'-phosphate, pantothenoylcysteine, pantetheine thiazoline, CoA).The metabolic role of pantethinase seems to be, in the light of the above results, connected with a degradative pathway of pantetheine-4'-phosphate, which is known to be one of the intermediates in the biosynthesis of CoA.A series of studies on the metabolism of cysteamine led to the identification of a new hydrolytic enzyme, which metabolises pantethine yielding pantothenic acid and cystamine, by cleavage of one of the carboamide bonds [l, 21. This enzyme, called pantethinase, is activated in the presence of cysteine (or other thiols as well), which increases enzymatic activity; nevertheless in the absence of any added thiol a little activity can be detected. To explain this behaviour two alternative hypotheses are possible : either the real substrate of the enzyme is the reduced form of pantethine or the enzyme requires the presence of reduced -SH groups in its molecule for fully catalytic activity. I n both cases the enzyme could show a little activity also in the absence of any activator, as actually observed, since the sulphidedisulphide exchange between the substrate molecule in the oxidized form and free -SH groups of the protein (or other contaminant proteins as well) could give rise to the active form(s). Of course, it can not be excluded a t all that both hypotheses are true a t the same time.

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This "malic enzyme particle" is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.

No 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...