Specific Activity Of Succinate Dehydrogenase Research Articles

The Effect of Initial Moisture Content, Packaging and Storage Period on Succinate Dehydrogenase and Cytochrome Oxidase Activity of Soybean Seed

The quality of soybean seed is easily decreased, so that it is difficult to keep them for a long time. The research aims to study the effect of initial moisture content, packaging material and storage period on succinate dehydrogenase and cytochrome oxidase activity. The experimental design was arranged in Factorial in RCB consisting of 3 factors: moisture content, i.e. 8. 10 and 12 percent, packaging materials, i.e. polyethylene, wheat and aluminium foil; storage period i.e. 0, 1, 2, 3, 4, 5, and 6 months. Changes in seed water content, specific activity of succinate and cytochrome oxidase, respiration rate, germination coefficient velocity germination, and abnormal seedlings were monthly determined. It was found that seed moisture content was increase and showed positive correlation with seed moisture content and abnormal seedlings and showed negative correlation with specific activity of succinate dehydrogenase and cytochrome oxidase, respiratory rate, germination and coefficient velocity germination. From this experiment, soybean seeds were stored in polyethylene and aluminium foil bags observed highly succinate dehydrogenase and cytochrome oxidase specific activity, respiration rate, germination, coefficient velocity germination and keep moisture content in low level could delay seed deterioration followed by polyethylene and wheat bags. Key words: moisture content, succinate dehydrogenase, cytochrome oxidase, soybean

The prevention of curcumin against rat liver mitochondrial swelling induced by tert-butylhydroperoxide

Liver diseases have been a medical problem which is difficult to manage. Some of the problems in the treatment of these diseases lie in the lack of reliable drug available. Curcumin, an active ingredient of the rhizomes of plant Curcuma has been investigated in the treatment of various disorders incuding liver diseases. The therapeutic effects of curcumin on liver diseases have been thought to be associated to its antioxidative properties. In the present study, we investigated the effects of curcumin on mitochondrial swelling in vitro induced by tert-butylhydroperoxide (t-BuOOH). Liver mitochondria were homogeneously isolated from Sprague-Dawley rats (the relative specific activity of succinate dehydrogenase was 35.73 ± 2.78). Addition of 90 µM of t-BuOOH caused a typical 2-phase swelling of the mitochondria. The pattern of swelling was influenced by various factors such as buffer composition, concentrations of t-BuOOH, amount of isolation buffer and mitochondrial proteins and incubation temperature.The swelling could be reduced by as much as 85 ± 3% by 2.50 µM of curcumin. At lower (1.25 µM) or higher (5.00 µM) concentrations, the protection against swelling by curcumin were less effective (respectively were 41 ± 3% and 77 ± 6%). Swelling might occur due to the opening of mitochondrial transition pore and could be an initial indication in the cascade process leading to cell death. The inhibition of t-BuOOH-induced mitochondrial swelling by curcumin might be because of the antioxidant effects of the compound. (Med J Indones 2006; 15:131-6) Keywords: mitochondria, swelling, tert-butylhydroperoxide, curcumin

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content.

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.

Fetal mitochondrial heart and skeletal muscle damage in Erythrocebus patas monkeys exposed in utero to 3'-azido-3'-deoxythymidine.

3'-azido-3'-deoxythymidine (AZT) is given to pregnant women positive for the human immunodeficiency virus type 1 (HIV-1) to reduce maternal-fetal viral transmission. To explore fetal mitochondrial consequences of this exposure, pregnant Erythrocebus patas monkeys were given daily doses of 1.5 mg (21% of the human daily dose) and 6.0 mg (86% of the human daily dose) of AZT/kg body weight (bw), for the second half of gestation. At term, electron microscopy of fetal cardiac and skeletal muscle showed abnormal and disrupted sarcomeres with myofibrillar loss. Some abnormally shaped mitochondria with disrupted cristae were observed in skeletal muscle myocytes. Oxidative phosphorylation (OXPHOS) enzyme assays showed dose-dependent alterations. At the human-equivalent dose of AZT (6 mg of AZT/kg bw), there was an approximately 85% decrease in the specific activity of NADH dehydrogenase (complex I) and three- to sixfold increases in specific activities of succinate dehydrogenase (complex II) and cytochrome-c oxidase (complex IV). Furthermore, a dose-dependent depletion of mitochondrial DNA levels was observed in both tissues. The data demonstrate that transplacental AZT exposure causes cardiac and skeletal muscle mitochondrial myopathy in the patas monkey fetus.

A biochemical evaluation of oral squamous cell carcinoma growth by measurement of specific activity of succinate dehydrogenase in the subrenal capsule assay

An auxiliary method for determination of chemosensitivity with the subrenal capsule assay (SRCA) was developed in which the specific activity of succinate dehydrogenase (SD) of tumor implanted beneath the renal capsule is measured. The appropriate conditions for measuring the specific activity of SD were determined. The chemosensitivity of tumors, derived from six xenograft lines originating from oral squamous cell carcinomas, to peplomycin (PEP), cis-platin (CDDP), and 5-fluorouracil (5-FU) were evaluated by the SRCA and the nude mouse assay (NMA). The chemosensitivity evaluated by NMA displayed a higher degree of correlation with that determined by the improved SRCA than with that determined by the conventional SRCA. The correlations between overall accuracy of prediction with the NMA and those with the conventional SRCA and the improved SRCA were 72.2% and 88.9%, respectively. These findings suggest that our new assay may be useful for evaluation of chemosensitivity in the SRCA.

コハク酸脱水素酵素比活性測定による制癌剤感受性試験（マウス腎被膜下移植法）の基礎的ならびに臨床的検討 宿主免疫反応の影響と臨床相関

Subrenal capsule assay (SRCA) is an in vivo chemosensitivity test which has been reported to be rapid and have high evaluability and clinical correspondence. However, chemosensitivity may be affected by infiltration of inflammatory cells derived from host imm une response and by poor tumor growth.We have developed an SRCA which was evaluated by measuring the specific activity of succinate dehydrogenase (SD). This novel method for evaluating SRCA was tentatively named the “SSDI method” because it was evaluated by determining the inhibition rate of specific activity of SD (SSDI) in a test group relative to a control group. In this investigation, fundamental and clinical studies on the SSDI method were carreid out.In SRCA, relative tumor weight (RW) and total activity of SD increased day by day, but the specific activity of SD (SSD) was almost constant.SSDI method was not influenced by the host immune response, compared with TGIR method.Furthermore, SRCA was carried out for 60 cases of malignant tumors in the oral region which were resected at our department. SSDI method showed a high evaluability rate (91.8%). For patients, the overall positive sensitivity rate was 42.2% by SSDI method and 28.6% by TGIR method. For drugs, the overall positive sensitivity rate was 25.4% by SSDI method and 17.3% by TGIR method. The positive sensitivity rates of anticancer agents evaluated by SSDI method were closer to the clinical sensitivity rates for malignant tumors in the oral region than those obtained by TGIR method. The predicitive accuracy rate was 73.9% by SSDI method and 52.2% by TGIR method.Therefore, SSDI method may be a useful assay to correctly evaluate chemosensitivity in SRCA.

Functional changes in rat-liver mitochondria during the early phase of burn injury

Using succinate as a substrate, the respiratory control ratios of liver mitochondria from burned male Sprague-Dawley rats with full skin thickness burns covering 20 per cent of the body surface area were increased at 15, 30, 45 and 60 min after burn, the peak being at 30 min post-burn. A sham group acted as control. In comparison with the sham group, the specific activity of succinic dehydrogenase was slightly decreased and the activities of cytochromes b and c+c were decreased significantly 30 min after burning. All the results suggest a lack of increased metabolic activity during the early phase of burn injury.

Metabolic Effects of Dietary Manganese Supplementation in ob/ob Mice

When fed a manganese-sufficient (20 ppm) diet, obese (ob/ob) mice have reduced levels of Mn in liver and brown adipose tissue (BAT), and depressed activities of succinate dehydrogenase (SDH) and manganese-containing superoxide dismutase (MnSOD) in BAT, compared to lean mice. Dietary Mn supplementation (200 ppm Mn) increased the Mn concentration in BAT in lean and obese mice and the Mn content of liver in the ob/ob mouse. Mn supplementation also led to an increase in the specific activities of SDH and MnSOD in BAT of lean and obese mice. In the obese mouse, these changes were paralleled by changes in the histological appearance of the tissue. The results indicate that the metabolism of Mn is altered in the liver and BAT of ob/ob mice, and that these alterations are responsive to dietary Mn supplementation.

Effects of commercial preparations of pangamic acid (B 15) on exercised rats

Exercise endurance and metabolic patterns were determined in rats that had been administered commercial preparations of “pangamic acid,” dimethylglycine, or diisopropylamine subcutaneously. None of these substances affected (a) the time required to reach exhaustion by treadmill running, (b) the specific activities of succinate dehydrogenase of liver and muscle, (c) the capacity of liver mitochondria for oxidative N-demethylation, (d) the content of mitochondria in liver, (e) the ratios of soluble to membranous protein of the liver mitochondria, or (f) the level of urinary excretion of creatinine. The concentrations of glycogen in muscle and liver were not increased. These results do not substantiate previous reports by Russian investigators on whose work many athletes base their practice of consuming pangamate to improve performance.

The activities in different neural cell types of certain enzymes associated with the metabolic compartmentation glutamate

The cellular distribution of certain enzymes associated with the metabolic compartmentation of glutamate was estimated in ultrastructurally preserved and metabolically competent perikarya fractions that were enriched in astrocytes, granule cells and Purkinje cells and derived from 8-day-old rat cerebellum, and in monolayer cultures (14 days in vitro) composed principally of interneurones or astrocytes. The neuronal activities of glutamine synthetase and glutamate dehydrogenase were respectively about 4- to 8-fold and 2- to 5-fold lower than in astrocytes, depending upon the class of neurone and the type of preparation used for comparison. By contrast glutaminase activity was about 3- to 12-fold higher in neuronal than in astroglial preparations. Estimations of the specific activity of succinate dehydrogenase differed less between cell types, indicating that the differences in glutamate dehydrogenase and glutaminase were not simply related to variations in the concentration of mitochondria relative to the other cellular constituents. The findings presented provide direct evidence in support of our model assigning the ‘small’ glutamate compartment, where most of the labelled glutamine is synthesized, to glial cells, and the ‘large’ compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.

Rough endoplasmic reticulum-mitochondrial complexes from rat liver: An extramitochondrial succinic dehydrogenase associated with this rough endoplasmic reticulum

The objective of this investigation was to determine if a mitochondrial inner membrane protein coded for by the nuclear genome could be identified with the rough endoplasmic reticulum associated with mitochondria. Rough endoplasmic reticulum-mitochondrial complexes were collected by zonal centrifugation. These associations were then subjected to hypotonic-hypertonic treatment and the mitoplasts were separated from rough endoplasmic reticulum and outer membranes. These were analysed for various marker enzymes including succinic dehydrogenase, succinate cytochrome c reductase, and cytochrome oxidase for inner mitochondrial membrane, monoamine oxidase for outer mitochondrial membrane, and glucose-6-phosphatase for rough endoplasmic reticulum. Some inner membrane damage occurred and contaminated the RER fraction as determined by cytochrome oxidase activity. However, the ratios of monoamine oxidase: cytochrome oxidase and glucose-6-phosphatase: cytochrome oxidase were over 20-fold and 105-fold greater, respectively, than for intact mitochondria indicating that inner mitochondrial membrane contamination was very low. Interestingly, succinic dehydrogenase activity was found to be present in the rough endoplasmic reticulum-outer mitochondrial membrane fraction, but succinate cytochrome c reductase activity was not detectable. In addition, the ratio of succinic dehydrogenase: cytochrome oxidase activity was 9-fold higher in the RER fraction than in mitochondria. These results were interpreted to mean that the succinic dehydrogenase activity in this fraction did not represent an enzyme which was a component of the electron transport chain. Specific activity of succinic dehydrogenase in the rough endoplasmic reticulum-outer mitochondrial membrane fraction was diminished in material from fasted rats and was increased in fasted-refed animals. Succinic dehydrogenase was also found localized on the rough endoplasmic reticulum by electron microscopic procedures using tetrazolium salts. These experiments support the hypothesis that succinic dehydrogenase is synthesized on the mitochondrially associated RER in preparation for transfer to the mitochondria.

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa 3 and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c 1, and aa 3, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.

Mitochondrial number, cytochrome oxidase and succinic dehydrogenase activity in Xenopus laevis oocytes

An estimate has been made of the numbers of mitochondria in the mitochondrial cloud (Balbiani body) of Xenopus laevis oocytes ranging in size from 50 to 250 micrometers. The mitochondrial number is expressed in terms of a 'standard' organelle measuring 2 micrometers in length and 0.2 micrometer in diameter and is derived by measurements on electron micrographs of sections through the cloud. It is found that the amount of mitochondrial material rises very rapidly as the oocyte grows in size. At the time the cloud disperses, in oocytes of about 300 micrometers in diameter, it is estimated that there are the equivalent of over 500 000 mitochondria in each cell. The rate of increase is very similar to the rate of accumulation of mitochondrial DNA during the same period of growth. Using a polarographic technique the specific activity of cytochrome oxidase and succinic dehydrogenase was determined in mitochondrial fractions isolated from oocytes over a size range of 80-1200 micrometers in diameter. Although the specific activity of succinic dehydrogenase remains constant that of cytochrome oxidase falls sharply during the period when the mitochondria are replicating rapidly, i.e. up to about 300 micrometer diameter. In larger oocytes the specific activity of enzymes appears to remain constant but increasing contamination of the isolated mitochondrial fraction does not allow conclusions to be drawn from the enzyme loading of the mitochondria once they have dispersed from the cloud. The results are discussed in relation to the possibility that mitochondrial replication preceeds, or at least outpaces, mitochondrial differentiation during the course of oogenesis.

Isolation of sarcolemma from newborn rabbit cardiac muscle

Sarcolemmal-enriched fractions from newborn cardiac muscles were isolated by three different methods and their purities were compared using morphological and biochemical techniques. The sarcolemmal-enriched fraction prepared using 0.6 m KCl extraction and sucrose gradient from newborn cardiac muscle contained high specific activities of K +-activated p-nitrophenyl phosphatase, Ca 2+-ATPase and (Na + + K +)-ATPase and low specific activities of succinate dehydrogenase, glucose-6-phosphatase, and NADPH cytochrome reductase. Electron microscopic examination of the sarcolemmal-enriched fraction prepared by the above method showed the presence of membranous vesicles and long segments of membranes and indicated high purity.

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K m , pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.

A comparative study on the composition of chlorosomes (Chlorobium vesicles) and cytoplasmic membranes from Chloroflexus aurantiacus strain Ok-70-fl and Chlorobium limicola f. thiosulfatophilum strain 6230

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.

Oxygen Uptake and Mitochondrial Enzyme Activities in the Mitotic Cycle of Physarum polycephalum

The rate of oxygen uptake by single macroplasmodia of Physarum polycephalum increased in two steps during each synchronous mitotic cycle. Plateaux in the respiratory pattern, of 0·22 cycles duration, occurred in mid-interphase and in the period up to and including mitosis. A fall in the rate of respiration was frequently associated with mitosis itself. This pattern of respiration continued for more than 10 mitotic cycles after inoculation and was unaffected by omission of an initial routine period of starvation. A similar stepped pattern of respiration was observed in synchronously germinating spherules during the period between outgrowth and the first mitosis. The specific activities of succinate dehydrogenase, fumarase and malate dehydrogenase remained relatively constant during the mitotic cycle, while fluctuations in cytochrome oxidase activity paralleled those in specific respiratory activity. Possible mechanisms for controlling the pattern of respiration are discussed with reference to published data on protoplasmic streaming and ATP concentrations during the mitotic cycle.

Effects of cateecolamine treatment as well as cold exposure on mitochondrial enzyme activities of the adipose tissue in a guinea-pig ( Cavia porcellus)

1. Catecholamine treatment at room temperature elevated the mitochondrial protein content and total activity of succinate dehydrogenase and cytochrome oxidase in adipose tissue of guinea-pigs, Cavia porcellus as did cold exposure, but changes were not so marked as in cold. 2. As with cold exposure the specific activity of cytochrome oxidase was increased but not that of succinate dehydrogenase by catecholamine treatment. 3. The specific activity of succinate dehydrogenase was, however, higher in the catecholamine-treated than in the cold-exposed animals. 4. The results show that exogenous catecholamine treatment had quite similar effects on the adipose tissue to those of cold, i.e. increased oxidative capacity of tissue.

Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle

Two populations of mitochondria were observed upon ultrastructural examination of cardiac muscle tissue, one located directly beneath the sarcolemma (subsarcolemmal mitochondria) and another between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria were released by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by nagarse digestion of the remaining tissue. These results were supported by electron microscopy of Polytron-treated heart tissue showing rupture and loss of sarcolemma with release of the underlying mitochondria but with retention of intact mitochondria between the myofibrils. Electron microscopy of the isolated mitochondria indicated that both mitochondrial types maintained their structural integrity throughout the isolation procedure. Specific activities of succinate dehydrogenase and citrate synthase were higher in the interfibrillar mitochondria as compared to the subsarcolemmal mitochondria, while those of carnitine palmitoyltransferase and alpha-glycerophosphate dehydrogenase were nearly the same in both. Interfibrillar mitochondria oxidized all substrates tested approximately 1.5 times faster than did the subsarcolemmal mitochondria. Thus the two mitochondrial types differed not only in their respective locations in the cell, but also in certain biochemical properties.

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.