The terminal step in the biosynthesis of L-ascorbic acid in peas has been shown to be the oxidation of L-galactono-y-lactone by an enzyme present in mitochondria (Mapson, Isherwood & Chen, 1954). This enzyme has been solubilized from mitocondria obtained from freshly germinated peas, cabbage leaves and cauliflower florets. Since the enzyme preparations from the mitochondria of the cauliflower florets were found to be more active than those from either peas or cabbage, we have used this material in most of the work reported here. The present paper describes the preparation and partial purification of the enzyme, together with an account of some of its properties. EXPERIMENTAL Ghemical8 L-Galactono-y-lactone was prepared by the reduction of D-galacturonic acid by borohydride as follows. D-Galacturonic acid (10 g.) was dissolved in 40 ml. of water and neutralized with NaOH to between pH 8-5 and 9 0. Borohydride was added gradually with stirring at room temperature. Samples were removed and acidified with acetic acid to remove excess of borohydride, and galacturonic acid was tested for by boiling with Fehling's solution. After reduction was complete, the solution was acidified with acetic acid to pH 5-0, a slight excess of barium acetate added, and the precipitate filtered off. Ethanol (2 vol.) was added to the solution and the precipitate was collected. After the precipitate had been washed twice with 60% (v/v) ethanol, barium was removed by Dowex 50 resin, and to the filtrate 1-2 drops of 3 N-HCl were added. The solution was concentrated to a syrup and dried in vacuo. The lactone was recrystallized from absolute ethanol.

Sutwumary. AMethionine can iniduce more thani a 100 % inlcrease in ethylene prodluction by apple tissue slices. The increased amounit of ethvlenie derives from carbolns 3 and 4 of methionine. On4l post-climacteric fruit tissues are stimulated bv iimethionine, and stimulatioin is optimum after 8 months' storage. Copper chelators such as sodium diethvl (lithiocarbam-nate and cuprizone very markedly inhibit ethylene production by tissuie slices. Carbon monoxide does not effect ethylene produiction by the slices. These (lata sluggest that the mechanism for the coniversion of mnethionine to ethylene, in apple tissues, is similar to the previously descriibed model system for pro(duicing ethylene fromii imiethionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systemis in tissues derives fromii miiethionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase. over inoninfiltrated treatmiienits, of rem,oving the preformiied ethylenie fromii the slices. Inl somiie later experiments air was replacedI by 0,. since it w-as found( that ethylene production by apple slices immiiiiersed in liquid media was stimulated b) O.-Gas Anialy-sis. Gases evolved by the tissue slices were sample(d by syringe anid determined by gas chromatography with a flamiie-ionization detector in a system using either alumiinia or silicolle (30% silicolne oil on celite) colulmniiis (3).T'racer EIxperinients. Tracer studies were carrie(d out with 14C. CH, labeled r-methionine, I)J.-methionine carboxy 4C, DL wlethioninle 2, 14C. or I)! n'ethionine 3, 4, 14C, added to the sucrose-bicarbonate inicubation mlixture. A 2 mil aliquiot of the gases evolved by the apple tissue slices xvas first assaye(l for ethylene onl the gas chromlatograph, and(I then 50 % of the gaseous atmosphere in the incuibation flask was remioved with a 50-nl gas-tight syringe for 14C analy sis.

1. A new reaction is described in which ethylene is formed from the Cu+-catalysed breakdown of methionine in phosphate buffer at 300 in air. Some of the other products of the reaction are methionine sulphone, methionine sulphoxide, homocysteic acid, homocystine, acrolein, dimethyl disulphide, methanethiol, ethyl methyl sulphide, methane and ethane. These are considered to be produced in different reaction pathways. The formation of ethylene in a model system in which Cu+ catalyses the breakdown of peroxidated linolenic acid has been described (Lieberman & Mapson, 1964). It was proposed that the biosynthesis ofethylene may proceed through an analogous system catalysed by a copper enzyme. Recent experiments, however, suggest that ethylene in tissues may arise from more than one source. For example, an analysis of particulate fractions, isolated from apples, shows that at least two types of compounds are present which can yield ethylene in the Cu+-catalysed system. The first is associated with the fat-soluble constituents of the cell and the second with the water-soluble constituents.Search for other precursors of ethylene has led to the finding that L-methionine, in reaction with the cuprous generating system, also forms ethylene. It is shown below that methionine and related substances are unique in their ability to form relatively large quantities of ethylene in a model system. The present paper describes characteristics of this ethylene-forming system, demonstrates that ethylene is derived from C-3 and C-4 of methionine, and suggests a mechanism for the reaction. MATERIALS AND METHODSModel 8y8tem. The model system consisted of L-methionine (1 mM), CU2+ (1.0 or 0-1 mm), ascorbate (10mm) and phosphate buffer, pH6.8 (60mm). Components of the 15 model system were contained in 25 ml. flasks sealed with one-hole rubber stoppers containing clamped capillary tubes. The flasks were incubated at 300 in a water bathshaker and internal atmospheres above the reaction mixture were sampled periodically with gas-tight syringes. Corrections were made to account for the increase in gas volume resulting from this sampling technique.Gas analysis. Gases produced in the reaction were determined by gas chromatography. Alumina or silicone (30% silicone oil on Celite) columns and a flame-ionization detector were used to determine ethylene. Details of the complete system have been described by Meigh, Norris, Craft & Lieberman (1960). Acrolein, ethyl methyl sulphide, dimethyl sulphide, dimethyl disulphide and methanethiol were detected on a capillary column (80ft. x 0-015in. diam.) at 150 with di-(2-cyanoethyl) ether as stationary phase, with an enrichment trap cooled in liquid 02 to first concentrate the gases. The vapours were subsequently released from the trap by passing an electric current through the trap.Methional. This compound was prepared from acrolein and methanethiol by the method described by Pierson, Giella & Tishler (1948