Yeast homogenate preparation

Whereas the structure and function of the 20S proteasome have been elucidated in great detail (for review see Baumeister et al 1998), the 19S regulator is understood only dimly at present. Structural studies are hampered by the low intrinsic stability of this assembly and extensive remodelling, which makes it notoriously difficult to obtain homogeneous preparations. Nevertheless, analyses of yeast. Drosophila and human 26S... 

Yeast Homogenate. Yeast homogenate (YH) was prepared by combining 5 g dried yeast with 25 g dry ice and grinding for 5 minutes with a mortar and pestle. The resulting yeast paste was taken up in 50 ml of water and kept on ice for immediate use or frozen at -20°C for future use. The YADH specific activity in YH varied between 5-10 U/mg protein. 

Animal tissue is a soiuce of papain-related lysosomal cysteine peptidases that have been used in di- and tripeptide synthesis. Due to the insufficient homogenic preparations of cathepsin B, earlier reports of its synthetic activity have to be taken witii caution [16]. The availability of a recombinant production system for hiunan cathepsin B will allow experiments with pure enzyme. The endopeptidase cathepsin L from parasite Fasciola hepatica was expressed in yeast and used in the kinetically controlled synthesis of Cbz-Phe-Arg-Ser-NH starting from Cbz-Phe-Arg-OMe and H-Ser-NH in an aqueous medimn [31]. 

Lennarz and Bloch (1960) have prepared 9-hydroxystearic acid-H from naturally occurring J -9-hydroxyoctadecenoic acid by catalytic hydrogenation, and racemic 10-hydroxystearic acid through epoxidation of oleic acid, and tested them as possible precursors to olefinic acids in two ways. Both were as efficient as oleic acid in satisfying the unsaturated fatty acid growth requirement of yeast raised under strictly anaerobic conditions, suggesting that they were desaturated to olefinic acids in vivo. 9-Hydroxystearic acid-H in addition was incubated with yeast homogenate in the presence of TPNH, ATP, and CoA, and yielded a monounsaturated acid with a double bond in the vicinity of C-8 to C-10. 

Mevalonic acid was discovered by Folker s group at Merck, Sharpe, and Dohme. The initial isolation was based upon the fact that it acted as a growth factor, or vitamin, for a strain of bacteria [35]. Once the structure had been determined, it was apparent that the molecule might well be the isoprenoid precursor that had been sought for many years. Subsequent experiments demonstrated that the sole (or nearly so) fate of the molecule was polyisoprenoid synthesis. In examining the role of cofactors necessary for the synthesis of cholesterol from mevalonate, only ATP and NADPH were found to be required. Experiments with a solubilized preparation from yeast demonstrated that there were 3 phosphorylated intermediates that could be isolated. These were shown to be mevalonic-5-phosphate, mevalonic-5-pyrophos-phate, and isopentenyl pyrophosphate [9]. These intermediates are derived from mevalonate in a sequence of phosphorylations, and the enzymes for all reactions have been obtained in homogeneous form. These enzymes, as well as the rest that lead to the synthesis of famesyl pyrophosphate, are cytosolic proteins. 

Another purified enzyme preparation which produces laminaripentaose from insoluble laminarin and from heat-treated pachyman is produced by a strain of Arthrobacter luteus (100,101,102) when grown on yeast cells or / -(1 —>3)-glucan. The enzyme, which was named Zymolase (also referred to as Zymolyase) appeared to be homogeneous by electrophoresis in a Tiselius apparatus and by ultracentrifugation. The molecular weight of the enzyme was estimated from ultracentrifugation to be ca. 20,500. The optimum pH for lysis of viable yeast cells was 7.5. The optimum temperature was 35°C. The optimum pH for heat-treated pachyman hydrolysis was 6.5, and the optimum temperature was 45°C. A Lineweaver-Burk plot with heat-treated pachyman yielded a Km value of 0.04% when the solubilized carbohydrate was assayed by the phenol-sulfuric acid method. Zymolase lost all its activity after incubation at 60°C for 5 min. 

This enzyme has been demonstrated in a range of plant tissues (Giovanelli and Mudd, 1967 Dodd and Cossins, 1969 Walker and Duerre, 1975) and has been partially purified from spinach beet (Poulton and Butt, 1976) and to homogeneity from yellow lupin seeds (Guranowski and Pawelkiewicz, 1977). The lupin preparation resembled the enzymes from rat liver (de la Haba and Cantoni, 1959) and yeast (Knudson and Yall, 1972) in showing high specificity for both L-homocysteine and adenosine. 

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