Yeasts cell-free extract

Schultz, M. C. (1999). Chromatin assembly in yeast cell-free extracts. Methods 17, 161—172. 

The isolation of 5-formyl-2-methylpyrimidine (62) and 5-amino-2-methylpyrimidine (63) from thiamin requiring mutants of Neurospora has led to a suggestion that these may be late intermediates in the biosynthesis of (45), a contention supported in part by the demonstration of conversion of the amino compound (63) to the hydroxymethylpyrimidine (45) by yeast cell-free extracts. 

Wohler s preparation of urea from ammonium cyanate, which could in principle be derived totally from inorganic constituents, is cited as an early demonstration (1828) that living cells were not obligatorily required for the synthesis of natural products. I can prepare urea without requiring a kidney or an animal—either man or dog. Three years after the death of Pasteur the finding by Hans and Edouard Buchner (1897) that fermentation still occured in a cell-free extract from yeast and so did not require the presence of organized cells, was virtually the final nail in the coffin for vitalism and an essential preliminary to the study of intermediary metabolism (Chapter 4). 

The same procedure was applied212 for the preparation of uridine 5 -(a-D-glucopyranosyl pyrophosphate-4"-t, or -5"-f. A cell-free extract of Phytophthora cinnamoni was used for the synthesis of the 6"-t derivative,213 as well as yeast pyrophosphorylase.2133 An enzyme from Salmonella typhimurium was found quite satisfactory214 for obtaining this and other labeled nucleotide sugars. ... 

The simplest example of an enzymic repair process is that of photoreactivation. This phenomenon was rediscovered in 1949 by Kelner, who found that UV-irradiated spores of Streptomyces showed higher survival if exposed to white light after the UV treatment (Kelner, 1949). At the time, this was not interpreted as an enzymic repair process but it was so. Enzymic action became demonstrable when it was shown that it could occur in vitro if, for example, transforming DNA of Haemophilus influenzae was incubated with cell-free extracts of yeast or E. coli after UV-treatment and then exposed to 400 nm light. (Rupert, 1958 1960). This assay made it possible to purify the enzyme from a variety of sources (e.g., Sutherland, 1974 Rupert, 1975). Once an assay for thymine dimers became available, it was shown that the action of the enzyme was to restore the dimers to monomeric thymidine in situ. 

The chemical nature of enzyme was controversial for a long time, until Buchner succeeded in isolating an enzyme system (zymase) from yeast in a cell-free extract in 1897.2) Urease was then crystallized by Sumner in 1926,3) followed by crystallization of several proteolytic enzymes by Northlop and his colleagues. At present the chemical nature of enzyme is defined as a protein with catalytic activity based on the specific activaiton of its substrate. However, this definition has been somewhat open to debate since a catalytic RNA, ribozyme, was discoved in 1982. 

Carbon-sulfur lyase, present in Eubacterium limosum cell-free extracts, can liberate volatile long-chain polyfunctional thiols, such as 4-MMP and 3-MH, from -cysteine conjugates. Gene knock-out and expression studies in yeast support the role of carbon-sulfur lyases (Tominaga et al. 1995 Howell et al. 2005 Swiegers et al. 2007). 

The conversion of glucose to ethanol demonstrated by a cell free extract from yeast. Buchner... 

COPII vesicles were first recognized when cell-free extracts of yeast rough ER membranes were incubated with cytosol, ATP, and a nonhydrolyzable analog of GTP. The vesicles that formed from the ER membranes had a distinct coat, similar to that on COPI vesicles but composed of different proteins, designated COPII proteins. Yeast cells with mutations in the genes for COPII proteins are class B sec mutants and accumulate proteins In the rough ER (see Figure 17-5). Analysis of such mutants has revealed several proteins required for formation of COPII vesicles. 

Arnold (51) partially purified such an enzyme from cell-free extracts of bakers yeast Matile et al, (45) and Cortat et al. (44) demonstrated the existence of glucanase-containing vesicles within the cytoplasm of Sac-charomyces cerevisiae. These vesicles contained exo- as well as endo-glucanases but the enzymes were not studied in detail. Fleet and Phaff (47) obtained qualitative evidence for the occurrence of endo-/ -( 1 — 3) glucanases in the cell walls of Saccharomyces rosei, Kluyveromyces fragilis, Hansenula anomala, Pichia pastoris, and Candida utilis. K. fragilis and H. anomala contained only exo-glucanase in cell extracts (38). 

Such universal distribution and distinct function does not mean that the enzymes could be readily studied everywhere. Due to their cell cycle dependence described later and rather poor activity in cell-free extracts ribonucleotide reductases can only be purified and characterized under carefully chosen conditions. Therefore the organisms named as enzyme source in this chapter are not too numerous. They include some obvious, biochemically well-known candidates for the study of DNA precursors like Escherichia coli, yeast, or calf thymus, while other, more remote ones (e.g., Brevibacterium ammoniagenes or the alga, Euglena gracilis) have been added because of some peculiarity of their DNA synthesis. A systematic view of enzymatic deoxyribonucleotide formation is assembled in Table 4. 

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