In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction nor produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by l-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55°C and had a pH optimum between 7–7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.     

Properties of the transfer ribonucleic acid methylase activity in vitro of hamster tumours induced by adenovirus-12. Base analysis

1. The ratio [ATP]/[ADP][P(i)], as measured by direct determination of the three components in rat liver, was found in various nutritional states to have approximately the same value as the ratio [ATP]/[ADP][P(i)] calculated from the concentrations of lactate, pyruvate, glyceraldehyde phosphate and 3-phosphoglycerate on the assumption that lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase are at near-equilibrium in the liver. This implies that the redox state of the NAD couple in the cytoplasm is linked to, and partially controlled by, the phosphorylation state of the adenine nucleotides. 2. The combined equilibrium constant of the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions at 38 degrees C and I0.25, was found to be 5.9x10(-6). 3. The fall of the [NAD(+)]/[NADH] ratio in starvation and other situations is taken to be the consequence of a primary fall of the [ATP]/[ADP][HPO(4) (2-)] ratio.     

Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts

Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF)-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G) and 5'-GGCGCC-3' (NarI site), induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.     

Lindane degradation by cell-free extracts of Clostridium rectum

For lindane degradation, a cell suspension of Clostridium rectum strain S-17 demands the addition of substrates such as leucine, alanine, pyruvate, a leucine-proline mixture, and molecular hydrogen. In the presence of leucine-proline mixture, lindane decomposed in parallel with isovaleric acid formation, and both lindane degradation and isovaleric acid formation were inhibited by monoiodoacetic acid, suggesting a close relation between lindane degradation and the Stickland reaction. Lindane was degraded by cell-free extracts of C. rectum in the presence of dithiothreitol (DTT). Radiogaschromatograms of n-hexane soluble metabolites from [¹⁴C] lindane showed the presence of monochlorobenzene and -3,4,5,6-tetrachlorocyclohexene, Leucine, NADH, and NADPH were somewhat less active than DTT for lindane degradation in cell-free extracts. Reductive dechlorination seemed the major route of lindane degradation in cell-free extracts as well as in the intact cells of C. rectum.Abbreviations Lindane (-HCH) -1,2,3,4,5,6-hexachlorocyclohexane - -HCH -1,2,3,4,5,6-hexachlorocyclohexane - -TCH -3,4,5,6-tetrachlorocyclohexene - -PCH -1,3,4,5,6-pentachlorocyclohexene - DTT 1,4-dithiothreitol     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Biosynthesis of mannoproteins by cell-free extracts fromPhycomyces blakesleeanus

Most of the manosyl transferase activity inPhycomyces blakesleeanus was found associated with a crude membrane fraction sedimenting at 48,400g (R_av). Triton X-100 and Nonidet NP-40 inhibited 95% of the enzyme activity. Digitonin caused 47% of inhibition and when removed, the membrane-bound enzymatic activity increased by about 35%; no activity was detected in supernatant. The rate of mannosyl transfer increased in the presence of 4 or 8 mM Mg²⁺ ions. Several compounds, including glycoproteins, mucoran, and mucoric acid, failed to act as acceptors of mannosyl residues. Guanosine diphosphate and guanosine monophosphate inhibited the transfer of mannosyl residues by 60 and 19%, respectively. Mannosyl transfer involves participation of lipid intermediates.β elimination of the product synthesizedin vitro revealed the presence of mannose, mannobiose, and mannotriose, suggesting that they are bound to protein viaO-glycosidic linkages. The alkaline-resistant carbohydrate part of the glycoproteins consisted mainly of mannose residues that were probably connected to the protein moiety throughN-glycosidic bonds.     

Methane oxidation by cell-free extracts of Methylococcus capsulatus

The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm).     

In vitro dehalogenation of tetrachloroethylene (PCE) by cell-free extracts of Clostridium bifermentans DPH-1

Cell-free extracts of Clostridium bifermentans DPH-1 catalyzed tetrachloroethylene (PCE) dechlorination. PCE degradation was stimulated by addition of a variety of electron donors. Ethanol (0.61 mM) was the most effective electron donor for PCE dechlorination. Maximum activity was recorded at 30 degrees C and pH 7.5. Addition of NADH as a cofactor stimulated enzymatic activity but the activity was not stimulated by addition of metal ions. When the cell-free enzyme extract was incubated in the presence of titanium citrate as a reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (0.5 mM). The activity of propyliodide-reacted enzyme was restored by illumination with a 250 W lamp. The dehalogenase activity was also inhibited by cyanide. The substrate spectrum of activity included trichloroethylene (TCE), cis-1,2-dichloroethylene (cDCE), trans-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloroethane, and 1,1,2-trichloroethane. The highest rate of degradation of the chlorinated aliphatic compounds was achieved with PCE, and PCE was principally degraded via TCE to cDCE. Results indicate that the dehalogenase could play a vital role in the breakdown of PCE as well as a variety of other chlorinated aliphatic compounds.     

Mycolic acid synthesis by Mycobacterium aurum cell-free extracts

The first cell-free system capable of synthesizing whole mycolic acids: (R1CH(OH)CH(R2)COOH, with 60 to 90 carbon atoms) from [1-14C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40 000 X g pellet) from disrupted cells of Mycobacterium aurum (ATCC 23366-type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolic acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When [1-14C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg2+ or Mn2+), KHCO3 and O2. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a carboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.