Enzymes In Cell-free Extracts Research Articles

Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli

Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell-free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37°C to 22°C.

Lactate Dehydrogenases in Obligately Anaerobic Chytridiomycetes from the Rumen

The enzyme D(-)lactate dehydogenase [D(-)lactate: NAD oxidoreductase, E. C. 1.1.1.28] has been detected in only a small num? ber of Chytridiomycetes, namely, Allomyces (Purohit and Turian, 1972), Blastocladiella (Rivedal and Sanner, 1978) and Blastocladia (Gleason and Gordon, 1989; Gleason and Price, 1969) which grow in the presence of oxygen and which are members ofthe order Blastocladiales. Orpin iso? lated and described three new genera of Chytridi? omycetes from sheep rumen, namely, Neocallimastix (Orpin, 1975), Piromonas (Orpin, 1976) and Sphaeromonas (Orpin, 1977) which are obligately anaerobic and which are members ofthe order Spizellomycetales (Heath et al, 1983). Similar obligately anaerobic fungi have been iso? lated from the rumen of other ruminants (see Phillips and Gordon, 1988). D(-)lactate, L(+)lactate, formate, acetate, ethanol, C02 and H2 have been reported to be products of fermentation of glucose in the three genera of rumen fungi (Bauchop and Mountfort, 1981; Phillips and Gordon, 1988; Borneman et al, 1989). Yarlett et al. (1986) assayed for a variety of enzymes in cell-free extracts from one isolate of Neocallimastix patriciarum and detected lactate dehy? drogenase but did not determine the specificity for the isomers of lactate. In this study we mea? sured the specific activities of lactate dehydro? genase as well as specificity of this enzyme for the isomers of lactate in cell-free extracts from six isolates of Neocallimastix, Piromonas and Sphaeromonas from the sheep rumen. The fungi were grown for four days at 39 C anaerobically on prereduced basal medium 10 plus glucose at a concentration of 5 g per liter. The complete composition of prereduced basal medium 10 is described by Phillips and Gordon (1988). Fungal mycelium was harvested by filtration on Mira cloth (Calbiochem, California) and washed with distilled water. The cell-free extract was prepared by grinding suspensions of mycelia in a solution containing 0.25 M sucrose and 0.01 M sodium phosphate buffer at pH 7.0 with a glass homogenizer or in the same solution with alumina in a mortar and pestle. During this procedure the temperature ofthe suspension was maintained near 0 C. The extract was clarified

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.

Accelerated ripening of Ras cheese with a commercial proteinase and intracellular enzymes from Lactobaciilus delbrueckii subsp bulgaricus, Propionibacterium freudenreichii and Brevibacterium linens

Summary - Intracellular cell-free enzyme extract of Lactobacillus delbrueckii subsp bulgaricus, Propionibacterium freudenreichii and Brevibacterium linens, was used to accelerate Ras cheese ripening with a commercial proteinase Neutrase. The general gross composition of the cheese was very similar during the ripening period. Significant increase in the free fatty acids of the cheese manufactured with the cell-free extract of Lactobacillus delbrueckii subsp bulgaricus was observed; protein breakdown showing little difference between the control and the treated cheese. After storage, the cheese treated with either Neutrase alone or with cell-free extract had significantly more intense typical Ras cheese flavour, but the presence of Neutrase and cell-free extract gave more bitterness at the end of ripening.

Temporal regulation of cdc2 mitotic kinase activity and cyclin degradation in cell-free extracts of Xenopus eggs

In cleaving Xenopus eggs, the cell division cycle is abbreviated to a rapid succession of S and M phases. During mitosis a number of proteins show increased phosphorylation due to the activation of a histone H1 kinase, the homologue of the cdc2+ gene product of the yeast Schizosaccharomyces pombe. We have studied the regulation of the activity of this enzyme in cell-free extracts of Xenopus eggs. In extracts of activated eggs incubated at 22 degrees C, histone H1 kinase activity shows two peaks of activation and disappearance. Activation occurs in two stages. The first stage requires protein synthesis, whereas the second does not. The second stage of activation involves post-translational activation of the kinase. Kinase activity rises to a peak and then abruptly disappears. Added sea urchin cyclin is degraded at the time of disappearance of kinase activity. The oscillation in kinase activity is then repeated, usually with lower amplitude. Post-translational activation of the kinase requires a membrane-containing particulate cellular component, whose role has yet to be defined. The kinase can still be activated in the presence of EDTA or in the presence of the ATP analogue, 6-dimethylaminopurine, which implies that phosphorylation of the kinase complex is not required for activation. Under these conditions, however, the kinase activity does not show its normal sudden disappearance, and added cyclin is perfectly stable. These observations are consistent with the idea that post-translational activation of the kinase involves protein phosphatase activity, whereas switching off the kinase requires an ATP-Mg2(+)-dependent reaction, perhaps due to protein phosphorylation

Cloning and identification of a cDNA from rat liver coding for a portion of L-gulonolactone oxidase.

An enzyme, l-gulono-γ-lactone oxidase, was purified from the livers of male albino rats and used to prepare a polyclonal rabbit antiserum. Reaction of the enzyme in cell-free extracts with the antiserum gave a single precipitin line when tested by the double diffusion method on agarose plates. Using the purified enzyme, the antiserum could detect nanogram quantities of the enzyme in dot-blotting procedures. This antiserum was used to screen a rat liver cDNA library in the vector λgtll. Positively reacting clones could easily be identified. One clone contained a cDNA insert of 800 bp and produced a β-galactosidase fusion protein with a MW 30,000 greater than the native molecule.

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H 2O 2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H 2O 2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H 2O 2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H 2O 2 concentration and/or time of exposure to H 2O 2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H 2O 2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H 2O 2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfyhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo proteins synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H 2O 2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics). We suggest that preferential degradation of H 2O 2-modified SOD may protect erythrocytes against the accumulation of oxidatively denatured SOD aggregates, and potentially toxic protein fragments.

Expression of degradative genes of Pseudomonas putida in Caulobacter crescentus.

The recombinant plasmid RP4-TOL was transferred into Caulobacter crescentus at a high frequency, and the plasmid was maintained for at least 50 generations. C. crescentus cells which contained RP4-TOL grew on all the aromatic compounds that the plasmid normally allowed Pseudomonas putida to grow on. Reciprocal transfers from C. crescentus donor to P. putida or Escherichia coli recipients were less efficient and occurred at frequencies of approximately 10(-3). Some representative TOL-specified enzymes in cell-free extracts of C. crescentus(RP4-TOL) were inducible, and their levels were similar to those of P. putida. The amounts of mRNA from induced cells of C. crescentus(RP4-TOL) and P. putida(RP4-TOL) were also similar. Moreover, the restriction enzyme digestion maps of RP4-TOL from both C. crescentus and P. putida were the same, indicating that the expression of the TOL genes occurred without any apparent alteration of the gene structure. This suggest that the degradative genes of Pseudomonas spp. can be transferred, maintained, and expressed efficiently in C. crescentus and that the mechanism of transcriptional activation of TOL genes observed in C. crescentus is similar to that of Pseudomonas spp.

Inhibitory effect of saccharin on glycolytic enzymes in cell-free extracts of Streptococcus mutans.

Inhibitory effect of sodium saccharin in concentrations from 10––5 to 10––2 M was tested on glycolytic enzymes involved in phosphate transfer in cell-free extra

Bacterial degradation of three dithioate pesticides

The bacterial metabolism of three dithioate pesticides; malathion, gusathion and dimethoate was examined in an effort to obtain cell-free enzyme systems for possible use in pesticide disposal and detoxification processes. Several bacterial strains from soil, sewage and sludge samples were isolated which utilized malathion and gusathion as their sole carbon sources whereas dimethoate was used only as a phosphorus source. Rapid metabolism of malathion and gusathion required strong agitation and aeration. In co-metabolite studies, pesticides disappeared more rapidly when methanol, ethanol, anthranilic acid, yeast extract or glucose were provided. Crude cell-free enzyme extracts obtained from these cultures hydrolyzed the three dithioate insecticides in the absence of co-factors. The enzymes appeared to be constitutive since the presence of dithioate compounds in the grown medium had no influence on the specific activity of the crude enzyme extracts.

Glutamine synthetase, glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH 4 (+) (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60-85%). The enzyme was adenylylated in bacteroids (43-75%) and in the nodule tissues (52-68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of (15)N-labelled (NH4)2SO4 into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on (15)N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.

Accelerated ripening of Cheddar cheese with a commercial proteinase and intracellular enzymes from starter streptococci

SUMMARYIntracellular cell-free enzyme extracts (CFE) of cheese starter bacteria significantly enhanced the flavour-accelerating effect of commercial neutral proteinase without increasing the gross proteolysis in Cheddar cheese. Peptide and amino acid N were released more rapidly in CFE-treated cheeses, compared with those treated with neutral proteinase or untreated controls.

Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide.

Approved type strains of Streptococcus sanguis, S. mitis, S. mutans, and S. salivarius were grown under aerobic and anaerobic conditions. The rate of hydrogen peroxide excretion, oxygen uptake, and acid production from glucose by washed-cell suspensions of these strains were studied, and the levels of enzymes in cell-free extracts which reduced oxygen, hydrogen peroxide, or hypothiocyanite (OSCN-) in the presence of NADH or NADPH were assayed. The effects of lactoperoxidase-thiocyanate-hydrogen peroxide on the rate of acid production and oxygen uptake by intact cells, the activity of glycolytic enzymes in cell-free extracts, and the levels of intracellular glycolytic intermediates were also studied. All strains consumed oxygen in the presence of glucose. S. sanguis, S. mitis, and anaerobically grown S. mutans excreted hydrogen peroxide. There was higher NADH oxidase and NADH peroxidase activity in aerobically grown cells than in anaerobically grown cells. NADPH oxidase activity was low in all species. Acid production, oxygen uptake, and, consequently, hydrogen peroxide excretion were inhibited in all the strains by lactoperoxidase-thiocyanate-hydrogen peroxide. S. sanguis and S. mitis had a higher capacity than S. mutans and S. salivarius to recover from this inhibition. Higher activity in the former strains of an NADH-OSCN oxidoreductase, which converted OSCN- into thiocyanate, explained this difference. The change in levels of intracellular glycolytic intermediates after inhibition of glycolysis by OSCN- and the actual activity of glycolytic enzymes in cell-free extracts in the presence of OSCN- indicated that the primary target of OSCN- in the glycolytic pathway was glyceraldehyde 3-phosphate dehydrogenase.

Enzymatic hydrolysis of malathion and other dithioate pesticides

Constitutive, cell free enzyme extracts derived from two strains of Arthrobacter sp. SB 3 and SB 4 hydrolyzed malathion and exhibited Km values of 1.3 and 2.0 μ mol/mg protein.min respectively. These two enzyme extracts had a broad pH optimum (6–9), a temperature optimum of 25 °C and 36 °C and were not strongly affected by high salt or solvent concentrations (up to 5%).

Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli.

The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli. Thus, nucleotide precursors labeled with [3H]diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation. Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol. Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains. The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined. At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide. Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously. Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis. This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA. CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells.

A cell-free system for Streptococcus faecium for studies on the biosynthesis of triterpenoid carotenoids.

A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.

The cryptic beta-fructofuranosidase of Saccharomyces rouxii.

The synthesis of beta-fructofuranosidase in synchronously dividing cells of S. rouxii was continuous (as opposed to periodic) throughout the budding cycle and followed the increase in cell mass. Similar patterns for cell mass and enzyme increases were observed even in phosphate-deprived cells which did not divide. The beta-fructofuranosidase activity remained physically cryptic throughout the cell cycle as evidenced by analyses on equilibrium density gradient fractions. The beta-fructofuranosidase activity released from mechanically disrupted cells resisted sedimentation when subjected to 131 000 g for 1 h, thus ruling out membrane association. Ethyl acetate was routinely employed to break the crypticity barrier. Enzyme in cell-free extract or in cells was equally sensitive to inactivation at pH values below 5 in the presence of ethyl acetate, which suggested that this is an inherent property of the enzyme in question and not a reflection of proteolytic inactivation. The status of beta-fructofuranosidase in selected species of Saccharomyces was compared with that for S. rouxii and a close similarity with S. bisporus var. mellis was noted. The degree of crypticity encountered in genetically defined strains of S. cerevisiae (e.g. X2180 a/alpha) was relatively high (42%) compared with that for commercially derived bakers' and brewers' strains (about 6%). Extant data on the cryptic beta-fructofuranosidase of S. rouxii are evaluated and the utility of this system for studying enzyme translocation is discussed.

Purification and structural properties of adenylylated and deadenylylated glutamine synthetase from Rhodopseudomonas sphaeroides

Glutamine synthetase (GS) of Rhodopseudomonas sphaeroides is regulated by adenylylation and deadenylylation. The extent of adenylylation/deadenylylation of the enzyme in cell free extracts was influenced by inorganic phosphate (Pi), α-ketoglutarate, ATP and other nucleotides. While Pi and α-ketoglutarate stimulated deadenylylation, ATP and other nucleotides enhanced adenylylation of the GS. By using proper combinations of the effectors and incubation conditions, any desired adenylylation state of GS could be adjusted in vitro. The enzyme was purified to electrophoretic homogenity by three steps including affinity chromatography on 5′-AMP-Sepharose. Adenylylated and deadenylylated enzyme showed different UV-spectra and isoelectric points. The native enzyme had a molecular weight of 600,000, deadenylylated subunits of 50,000±1,000. Electron microscopic investigations revealed a dodecameric arrangement of subunits in two hexameric planes.

Adenine and adenosine metabolizing enzymes in cell-free extracts from Euglena gracilis

1. 1. Activities of the following enzymes involved in adenine and adenosine metabolism were found in cell-free extracts from Euglena gracilis: acid phosphatase (EC 3.1.3.2), 5′-methylthioadenosine phosphorylase (EC 2.4.2.-), adenine deaminase (EC 3.5.4.2), adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). 2. 2. The activities occurred both in heterotrophic and photoautotrophic cells and their levels did not change during light-induced chloroplast development. 3. 3. Neither S- adenosylhomocysteinase (EC 3.3.1.1), 5′-methylthioadenosine nucleosidase (EC 3.2.2.9) and nucleoside phosphotransferase (EC 2.7.1.77) nor adenosine degrading enzymes: adenosine deaminase (EC 3.5.4.4), adenosine nucleosidase (EC 3.2.2.7), and purine-nucleoside (adenosine) phosphorylase (EC 2.4.2.1) were found in the Euglena extracts. 4. 4. Comparison of the adenine and adenosine metabolism in Euglena and in other organisms is comprehensively presented. The metabolism in Euglena gracilis differs from that in higher animals and plants.

Ethane oxidation by methane-oxidizing bacteria.

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible. Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the moo-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.