Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors.

This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23-0.99 U mg(-1) protein), butyrate kinase (Buk, < 0.03 U mg(-1) protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24-7.64 U mg(-1) protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH3 and NH4(+)-N), and a negative dependency can be postulated. Thus, high concentrations of NH3 and NH4(+)-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities.

Monitoring succinyl-CoA:3-oxoacid CoA transferase nitration in mitochondria using monoclonal antibodies.

Two tyrosine residues (Tyr(4) and Tyr(76)) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr(4) or Tyr(76) of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr(4) and Tyr(76) in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.

Assaying for the 3-hydroxypropionate cycle of carbon fixation.

The 3-hydroxypropionate cycle is a novel pathway for autotrophic CO2 fixation, which has been demonstrated in the thermophilic phototrophic bacterium Chloroflexus aurantiacus; a yet to be defined variant of this pathway occurs in autotrophic members of the Sulfolobales (Crenarchaeota). The 3-hydroxypropionate cycle consists of the conversion of acetyl-CoA to succinyl-CoA, via malonyl-CoA, 3-hydroxypropionate, propionyl-CoA, and methylmalonyl-CoA. Carboxylation of acetyl-CoA and propionyl-CoA by acetyl-CoA/propionyl-CoA carboxylase are the CO2 fixation reactions. Succinyl-CoA serves as a precursor of cell carbon and also as a precursor of the starting compound acetyl-CoA. In C. aurantiacus, the cycle is completed by converting succinyl-CoA to malyl-CoA and cleaving malyl-CoA to acetyl-CoA and glyoxylate. Glyoxylate is then converted in a second cyclic pathway to pyruvate, which serves as a universal cell carbon precursor. The fate of succinyl-CoA in Sulfolobales is at issue. Assays used to study the characteristic enzymes of this novel pathway in C. aurantiacus are reported.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine.

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.

Acyl-CoA transferase activities in homogenates of Fasciola hepatica adults.

Succinyl-CoA is an intermediate in the formation of the fermentation product, propionate, by Fasciola hepatica adults. Acyl-CoA transferase activities are present in crude homogenates of Fasciola, which could account for the synthesis of succinyl-CoA from succinate by the transfer of CoA from either propionyl-CoA or acetyl-CoA. No transferase activity was apparent from 2-methylbutyryl-CoA or 2-methylvaleryl-CoA as was previously reported for the nematode, Ascaris suum. Heat denaturation experiments indicate that all of the Fasciola transferase activities may result from a single protein.

Prenatal diagnosis of succinyl-coenzyme A:3-ketoacid coenzyme A transferase deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare disorder of ketone body catabolism. In the present study, we prenatally diagnosed SCOT deficiency in a fetus in a family of which the proband was the first patient with SCOT deficiency identified in Japan, by analysis of enzyme activity levels in samples of chorionic villi and cultured amniocytes. In the fetus of the family, SCOT activity was not detected in either chorionic villi or cultured amniocytes. Since the levels of SCOT activity in control chorionic villi were close to our minimal detectable level and were much lower than those in control cultured amniocytes, enzyme assay in cultured amniocytes was more feasible than that in chorionic villi for prenatal diagnosis of SCOT deficiency. No elevated accumulation of 3-hydroxybutyrate or acetoacetate was detected in the amniotic fluid of the fetus. To our knowledge, this report is the first of prenatal diagnosis of SCOT deficiency.

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2.

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.

Growth and development of brain of artificially reared hypoketonemic rat pups.

Three groups of rats were reared: mother-reared controls; artificially reared controls (AR-c), which were fed a milk substitute with the same composition of macro-nutrients as natural rat's milk; and an artificially reared test group (AR-h), which was fed a milk substitute identical to that fed AR-c pups except that the component of fat containing medium chain length fatty acids was omitted (medium chain triglyceride deficient) and replaced on an isocaloric basis with carbohydrate. The AR rats were fed the milk substitute from postnatal Day 5 until Day 17 by fitting them with gastric cannulas through which the milk could be infused automatically. The nutritional impact of the milk substitutes on growth and the integrity of the brain was assessed by a comparison of morphologic and biochemical markers. Pups in the AR-h group were hypoketonemic. Animals in all groups attained the same body weight by Day 17 and there was no difference in the morphologic markers among the groups with one exception: the vibrissal "barrel fields" of the somatosensory cortex of rat pups in both AR groups were reduced in size but not in number of distribution from those of the mother-reared groups. Furthermore, the brains of the rat pups in the AR groups were not different in weight, but they weighed less than brains of mother-reared controls. Our data show that although there are many similarities in the status of AR rat pups when compared with mother-reared controls, distinctive differences associated with artificial rearing are evident. We conclude that medium chain fatty acids in milk fat and the circulating ketone bodies are not mandatory substrates for growth and the development of the brain. Mechanisms must exist whereby alternative substrates are used to compensate when these metabolites are diminished in supply.

Regulation of succinyl coenzyme A:acetoacetyl coenzyme A transferase in rat hepatoma cell lines.

The regulation of succinyl-CoA:acetoacetyl-CoA transferase (CoA transferase) has been studied in 8 rat hepatoma cell lines. Compared with normal rat hepatocytes, which have almost nondetectable activity of the enzyme, the hepatoma cell lines have a wide range of expression of CoA transferase activity, from as low as 45 nmol/min/mg to as high as 960 nmol/min/mg. Western blotting showed that the different levels of CoA transferase activity were due to differing amounts of the enzyme in the cells. This was further attributed to the varying amounts of the enzyme synthesized in the cells as monitored by L-[35S]methionine labeling followed by immunoprecipitation. To study further the differential expression of CoA transferase in the hepatoma cell lines, the relative quantity of functional CoA-transferase mRNA in the cells was measured by in vitro translation. The results showed that the levels of functional CoA transferase mRNA detected were consistent with the differences in the enzyme activity in the cells. Since CoA transferase is the key enzyme responsible for the utilization of ketone bodies as an alternative energy source, the expression of CoA transferase in hepatoma cells may play a role in energy production.

The effects of dietary fiber feeding on cholesterol metabolism in rats.

The flux through the sterol biosynthetic pathway was studied in hepatocytes isolated from male Sprague-Dawley rats fed diets containing one of four fiber sources: cellulose, pectin, oat bran and wheat bran. Sterol synthesis measured by the incorporation of tritiated water or [2-14C]mevalonic acid was not inhibited in hepatocytes isolated from animals fed diets containing cellulose, pectin, oat bran or wheat bran when compared to animals fed a fiber-free diet. Based on these results, it is concluded that the intake of fiber has no inhibitory effect on endogenous sterol synthesis. In fact, in comparison to that in fiber-free controls, sterol synthesis was markedly elevated in pectin- and wheat bran-fed animals. In the case of the pectin-treated animals, the higher synthetic rate corresponded to an increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Regulation of D-beta-hydroxybutyrate dehydrogenase in rat hepatoma cell lines.

Quantitation of D-beta-hydroxybutyrate dehydrogenase (BDH) in normal rat hepatocytes was compared with that in two rat hepatoma cell lines, H4-II-EC3 and RLT-3C. BDH activity in normal rat hepatocyte mitochondria was 321 nmol/min/mg, which was greatly reduced to 10.7 nmol/min/mg and 1.7 nmol/min/mg in H4-II-EC3 and RLT-3C cell mitochondria, respectively. The cell growth rate and L-[35S]methionine incorporation rate showed that RLT-3C cells had the highest growth rate (32.4-h doubling time) and the fastest protein biosynthesis rate (2.65 x 10(5) cpm/min/10(6) cells). The H4-II-EC3 cell line grew more slowly (48.5-h doubling time) and had lower protein biosynthesis rate (1.46 x 10(5) cpm/min/10(6) cells). The protein synthesis rate in hepatocytes was 1.25 x 10(5) cpm/min/10(6) cells. These results suggest that there is a reciprocal correlation between BDH activity and cell growth and protein synthesis rates. Immunochemical quantitation of BDH showed the amount of BDH in H4-II-EC3 and RLT-3C cells was about 4.8 and 0.5% of that in normal rat hepatocytes, respectively. Quantitation of BDH by biosynthesis indicated that BDH content in H4-II-EC3 cells and RLT-3C cells was 9.3 and 4.0% of that of normal hepatocytes, respectively. Precursor BDH synthesized by in vitro translation primed with RNA of H4-II-EC3 cells or RLT-3C cells was 3.0 and 1.1% of that translated from normal rat hepatocyte RNA. These results suggest that the decrease in BDH content in hepatoma cells results from a decrease in functional BDH-mRNA. The coupling of a decrease in BDH activity with an increase in activity of succinyl-CoA: acetoacetyl-CoA transferase in hepatoma cells may play a role in generating additional energy required for the rapid growth of tumor cells.