The S-n-propyl analogue of S-adenosylmethionine.

A special strain of Saccharomyces cerevisiae responded to a supplement of S-n-propyl-L-homocysteine in the culture medium by synthesizing S-adenosyl-(S-n-propyl)L-homocysteine, the S-n-propyl analogue of S-adenosylmethionine. S-n-Butyl-L-homocysteine reacted sparingly with this strain, but S-isopropyl-L-homocysteine failed to form detectable quantities of the corresponding S-adenosylsulfonium compound. The S-n-propyl compound was isolated by extraction of the cells, followed by ion-exchange chromatography, which separated it from endogenous S-adenosylmethionine. The structure was determined by hydrolytic procedures leading to overlapping fragments of known structure, 5'-n-propylthioadenosine and S-n-propyl-L-homocysteine. The new sulfonium compound was examined for its activity as n-propyl donor by substituting it for S-adenosylmethionine in methyltransferase systems. Enzymatic transpropylation was observed with S-adenosylmethionine : L-homocysteine S-methyltransferase (EC 2.1.1.10). Its rate was low in the S-adenosylmethionine : N-acetylserotonin O-methyltransferase system (EC 2.1.1.4), and below recognition with S-adenosylmethionine : guanidinoacetate methyltransferase (EC 2.1.1.2) and S-adenosylmethionine : histamine N-methyltransferase (EC 2.1.1.8).

Properties of yeast cell ghosts obtained by ribonuclease action.

Cell ghosts were obtained from Candida utilis and Saccharomyces cerevisiae by the action of some conformational isomers of pancreatic ribonuclease which passed through the cell wall and penetrated the cell membrane. In the interior, the enzyme caused extensive disorganization of the cellular structure as evidenced by the results of vital staining and electron microscopy. Ribonucleic acid was degraded to fragments that leaked out into the suspending medium. Other cytoplasmic constituents, including the amino acid pool, were released, but most of the cellular protein and deoxyribonucleic acid remained in the ghosts. The lesions in the cytoplasmic membrane were too small to be seen by conventional electron microscopy. The membrane clung to the cell wall even after obliteration of most of the intracellular structure.

Effect of some proteins on the yeast cell membrane.

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.

Ultraviolet micrography of penetration of exogenous cytochrome c into the yeast cell.

Candida utilis and Saccharomyces cerevisiae in water suspension were found to be very sensitive to exogenous cytochrome c. The protein was taken up by the cells, and the viable count was reduced to a few per cent of the initial value. Micrography at 405 nm revealed penetration of cytochrome c into the interior of the cell. The cytoplasmic membrane lost its capacity to retain intracellular constituents, and ultraviolet-absorbing compounds were released into the medium. When budding cells were subjected to treatment with cytochrome c, the mother cells were found to be more susceptible than the buds. Phosphate buffer protected the cells and spheroplasts against cytochrome c.