Lower Citrate Synthase Activity Research Articles

H55N polymorphism as a likely cause of variation in citrate synthase activity of mouse skeletal muscle

Citrate synthase (CS) is an enzyme of the Krebs cycle that plays a key role in mitochondrial metabolism. The aim of this study was to investigate the mechanisms underlying low activity of citrate synthase (CS) in A/J mice compared with other inbred strains of mice. Enzyme activity, protein content, and mRNA levels of CS were studied in the quadriceps muscles of A/J, BALB/cByJ, C57BL/6J, C3H/HeJ, DBA/2J, and PWD/PhJ strains of mice. Cytochrome c protein content was also measured. The results of the study indicate that A/J mice have a 50-65% reduction in CS activity compared with other strains despite similar levels of Cs mRNA and lack of differences in CS and cytochrome c protein content. CS from A/J mice also showed lower Michaelis constant (K(m)) for both acetyl CoA and oxaloacetate compared with the other strains of mice. In silico analysis of the genomic sequence identified a nonsynonymous single nucleotide polymorphism (SNP) (rs29358506, H55N) in Cs gene occurring near the site of the protein interacting with acetyl CoA. Allelic variants of the polymorphism segregated with the catalytic properties of CS enzyme among the strains. In summary, H55N polymorphism in Cs could be the underlying cause of low CS activity and its high affinity for substrates in A/J mice compared with other strains. This SNP might also play a role in resistance to obesity of A/J mice.

Abnormal cardiac morphology, function and energy metabolism in the dystrophic mdx mouse: An MRI and MRS study

Patients with muscular dystrophy have abnormal cardiac function and decreased high-energy phosphate metabolism. Here, we have determined whether the 8 month old mdx mouse, an animal model of muscular dystrophy, also has abnormal cardiac function and energetics. In vivo cardiac MRI revealed 33% and 104% larger right ventricular end-diastolic and end-systolic volumes, respectively, and 17% lower right ventricular ejection fractions in mdx mice compared with controls. Evidence of left ventricular diastolic dysfunction included 18% lower peak filling rates in mdx mouse hearts. Abnormal cardiac function was accompanied by necrosis and lower citrate synthase activity in the mdx mouse heart, suggesting decreased mitochondrial content. Decreased mitochondrial numbers were associated with 38% lower phosphocreatine concentration, 22% lower total creatine, 36% higher cytosolic free ADP concentration and 1.3 kJ/mol lower free-energy available from ATP hydrolysis in whole isolated, perfused mdx mouse hearts than in controls. Transsarcolemmal creatine uptake was 12% lower in mdx mouse hearts. We conclude that the absence of dystrophin in adult mdx mouse heart, as in the heart of human patient, is associated with right ventricular dilatation, left ventricular diastolic dysfunction and abnormal energy metabolism.

Relationship of physical function to vastus lateralis capillary density and metabolic enzyme activity in elderly men and women

There are no data showing whether or not age-related declines in physical function are related to in vitro properties of human skeletal muscle. The purpose of this study was to determine whether physical function is independently associated with histologic and metabolic properties of skeletal muscle in elderly adults. The study was a cross-sectional observational study of 39 sedentary, older (60-85 yrs) men and women. A needle biopsy of the vastus lateralis for assessment of muscle fiber type, fiber area, capillary density and citrate synthase and aldolase activities was performed. Physical function tests included the Short Physical Performance Battery (balance, walking speed, and chair rise time), as well as self-reported disability. Total fiber area (R=-0.41, p=0.02), number of Type II fibers (R=-0.33, p=0.05), and aldolase activity (R=-0.54, p=0.01) were inversely related to age. Persons who reported greater difficulty with daily activities had lower capillary density (R=-0.51, p=0.03) and lower citrate synthase activity (R=-0.66, p=0.03). Walking speed was directly related to fiber area (R=0.40, p=0.02), capillary density (R=0.39, p=0.03), citrate synthase (R=0.45, p=0.03) and aldolase (R=0.55, p<0.01) activities, even after adjustment for age, BMI and disease status. In older adults, skeletal muscle capillary density and metabolic enzymatic activity are independent predictors of lower extremity physical function.

Thyroid status and nitric oxide in rat arterial vessels

Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively. We therefore hypothesized that the capacity for vascular nitric oxide (NO) formation is decreased in hypothyroidism and increased in hyperthyroidism. To test this hypothesis, rats were made hypothyroid (HYPO) with propylthiouracil or hyperthyroid (HYPER) with triiodothyronine over 3-4 months. Compared with euthyroid control rats (EUT), HYPO exhibited blunted growth and lower citrate synthase activity in the soleus muscle; HYPER exhibited left ventricular hypertrophy and higher citrate synthase activity in the soleus muscle (P<0.05 for all effects). The capacity for NO formation was determined in aortic extracts by formation of [3H]L-citrulline from [3H]L-arginine, i.e. NO synthase (NOS) activity. Thyroid status modulated NOS activity (EUT, 36.8 +/- 5.5 fmol/h per mg protein; HYPO, 26.0 +/- 7.9; HYPER, 64.6 +/- 12.7; P<0.05, HYPER vs HYPO). Expression of endothelial and neural isoforms of NOS was modulated by thyroid status in a parallel fashion. Capacity for responding to NO was also determined via measuring cGMP concentration in aortae incubated with sodium nitroprusside. Stimulated cGMP formation was also modulated by thyroid status (EUT, 73.0 +/- 20.2 pmol/mg protein; HYPO, 152.4 +/- 48.7; HYPER, 10.4 +/- 2.6; P<0.05, HYPER vs HYPO). These data indicate that thyroid status alters capacities for both formation of and responding to NO. The former finding may contribute to previous findings concerning vascular function in thyroid disease states.

A novel enzyme, citryl-CoA lyase, catalysing the second step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6.

A novel enzyme catalysing citryl-CoA cleavage to acetyl-CoA and oxaloacetate was purified from Hydrogenobacter thermophilus TK-6, and designated citryl-CoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i). citryl-CoA formation by citryl-CoA synthetase (CCS) and (ii). citryl-CoA cleavage by CCL. Purified CCL gave a single 30 kDa band in SDS-PAGE and gel filtration chromatography indicated that the native state of the enzyme exists as a trimer (alpha(3)). Citryl-CoA lyase showed low citrate synthase (CS) activity. Using an oligonucleotide probe, the corresponding gene was cloned and sequenced. The gene was expressed in Escherichia coli and recombinant CCL was also purified. The CCL protein sequence showed similarity to the C-terminal regions of ATP citrate lyase (ACL) and CS sequences in the database. By further sequence comparisons, the phylogenetic relationship between CCS, CCL, ACL and CS was investigated.

Seasonal variation in biochemical indicators of physiological status in Euphausia superba from Port Foster, Deception Island, Antarctica

Seasonal changes in biochemical indicators of physiological status were analyzed in abdominal muscle of the Antarctic krill, Euphausia superba, collectedfrom Port Foster, Deception Island , an active volcano locatedin the ShetlandIslandchain west of the Antarctic Peninsula. Krill were collectedwith a 10 m 2 MOCNESS trawl during four cruises (November 1999, February, May, November 2000). RNA:DNA mirroredthe chlorophyll a concentration, with the highest values found during seasons of abundant phytoplankton. Activities of the glycolytic enzyme lactate dehydrogenase (LDH) and the mitochondrial enzyme citrate synthase (CS) were significantly higher in male krill when comparedto females of similar size, indicating that their burst andaerobic swimming performance may be higher than females throughout the year. RNA:DNA ratio andenzyme activities were highly elevatedin summer as comparedto the earliest spring sampling period. Krill showed significant seasonal changes in LDH activity, with lowest values in spring andhighest values in summer (females) or autumn (males). Krill showedsignificant seasonal changes in CS activity with highest values in summer. Protein and% water variedsignificantly among seasons for both males and females. Lower CS activity and RNA:DNA ratio suggest krill exhibit reduced metabolism during the winter when phytoplankton production is reduced, perhaps enhancing survival. Lower enzyme activities in female krill in early spring suggest they may achieve greater metabolic suppression during overwintering. r 2003 Elsevier Ltd. All rights reserved.

Metabolic properties of Northern krill, Meganyctiphanes norvegica , from different climatic zones. II. Enzyme characteristics and activities

Activities and characteristics of two metabolic key enzymes, citrate synthase (CS) and pyruvate kinase (PK), were studied in the Northern krill, Meganyctiphanes norvegica, with respect to adaptive properties under different thermal conditions. Krill were sampled during late winter/spring and summer from the constantly warm Ligurian Sea (12–13°C below the thermocline), the colder but also comparatively constant Clyde Sea (7–8°C), and the variable Kattegat (2–16°C). Both enzymes showed distinct tissue- and organ-specific activities, which were highest in the pleopods – the principal locomotive organs. The fourth and fifth abdominal segments, however, were used for routine investigation due to lowest variability. Specific activity of CS and PK did not differ between seasons in krill from the Kattegat or the Clyde Sea. In the Ligurian Sea, in contrast, specific CS activities were significantly lower during summer. Analysis of individual data illustrated a decrease of CS activity with size and an increase of PK activity with size. Taking these allometric effects into account, as emphasized by calculating the ratio between both enzymes, variation of CS and PK activities turned out to be solely dependent on body size, which differed between locations and seasons. Ligurian krill from the summer, however, were unique in that they showed a lower CS/PK ratio than would be predicted by the scaling effect. Thermal characteristics of each enzyme were similar between locations and seasons. During the winter, in Kattegat and Clyde Sea krill, K m values (Michaelis–Menten constant) of CS towards acetyl-coenzyme A exhibited an almost constant level over the experimental temperature range of 4–16°C. During summer, however, K m values were lower at 8°C in the Clyde Sea and at 12°C in the Kattegat. In Ligurian krill from the summer, K m values were consistently lower than those of winter krill over the entire experimental temperature range. In conclusion, Kattegat and Clyde Sea krill show only minor adaptations to their respective thermal environments in terms of CS and PK characteristics. Ligurian krill, in contrast, exhibited decreased specific CS activity during summer, which might be compensated by elevated enzyme–substrate affinity as indicated by lower K m values. Since temperature was constant during both seasons, this effect cannot be explained as a reaction to thermal conditions. Consequently, oligotrophic conditions in the Ligurian Sea during summer may entail a reduction in the somatic performance of krill, which is reflected by lower CS activity.

DECREASED PHYSICAL ACTIVITY BLUNTS ACETYLCHOLINE-INDUCED INCREASES IN RAT SKELETAL MUSCLE BLOOD FLOW

593 The purpose of this study was to examine the effects of decreased muscle activity due to hindlimb unweighting on endothelium-dependent control of skeletal muscle blood flow (BF) in rats. We hypothesized that, compared to casted control (CC) rats, hindlimb unweighted (HU) rats would exhibit blunted increases in BF during infusions of acetylcholine (ACh) into the hindlimb vasculature. HU (n=9) rats were suspended for 13-14 days. Efficacy of decreased muscle activity was indicated by lower citrate synthase activity in Gr and Gw (CC vs HU: 33.3±1.1 vs 22.3±2.6 and 10.5±0.7 vs 8.6±0.7 μmol/min/g, p < 0.04) and P (20.0±1.0 vs 16.9±1.5, p = 0.051). After anesthetizing the rat (Inactin 100 mg/kg ip), ACh was infused into the left femoral artery via the epigastric artery. Regional muscle BF to the left leg (soleus (S), plantaris (P), and red (Gr), white (Gw), and mixed (Gm) gastrocnemius) was measured using radiolabeled microspheres before and during ACh infusions of 0.2-0.4 and 1.4-2.0μg/kg/min (BF-high). The BF response to ACh (BF-high) was significantly decreased by HU in P (CC vs HU: 19.2±3.6 vs 7.9±0.9 ml/min/100g, p < 0.02) and Gw (CC vs HU: 9.4±1.5 vs 4.0±0.8 ml/min/100g, p< 0.03), but not S, Gr, or Gm muscles. Mean arterial pressure did not differ between groups (p > 0.10), although there was a significant dose effect of ACh infusion rate (p < 0.01). We conclude HU diminishes ACh induced increases in muscle BF to the P and Gw, possibly through a blunted endothelium response.

Aerobic glucose fermentation by Trypanosoma cruzi axenic culture amastigote-like forms during growth and differentiation to epimastigotes

Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28°C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH 3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH 3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.

Carnitine acyltransferases and acyl-CoA hydrolases in human and rat liver.

The activities of carnitine acyltransferases and acyl-CoA hydrolases were determined in human and rat liver to establish the validity of extrapolating from studies on rats to human metabolism. In human liver, carnitine acetyltransferase activity was 10-14 times higher and carnitine octanoyltransferase 1.7-2.4 times higher than in rat liver, while carnitine palmitoyltransferase activity was similar in human and rat. Acetyl-CoA hydrolase and octanoyl-CoA hydrolase activities were lower in human (42-57%) than in rat liver, but palmitoyl-CoA hydrolase activity was similar in both species. The activity of citrate synthase was lower (44%) in human than in rat liver. The low citrate synthase activity and the high carnitine acetyltransferase in human liver suggest that in man acetylcarnitine might be more important as a vehicle for export of acetyl units from mitochondria than citrate. The high activity of carnitine acetyltransferase in human liver is consistent with the observation that acetylcarnitine is the predominant acylcarnitine excreted in diabetic ketosis in man. It is concluded that the rat may not be a valid model for carnitine metabolism in man, and that in human liver carnitine may have an important role in transfer of acetyl groups out of mitochondria and possibly also to extra-hepatic tissues.

Production of aspartic acid and lysine by citrate synthase mutants of Brevibacterium flavum.

A prototrophic revertant was derived from a citrate synthase-defective glutamate auxotroph of Brevibacterium flavum. It showed low citrate synthase activity and overproduced L-aspartic acid. The maximum production was 10.6 g/liter (about a 30% yield) when the strain was cultured in medium containing 36 g/liter of glucose for 48 hr. The amount of aspartic acid produced depended greatly on the concentration of biotin in the medium, as the case of glutamate production by the original wild strain. When the revertant strain was cultured with excess biotin, acetic acid accumulated instead of aspartic acid. A mutant resistant to S-(2-aminoethyl)-L-cysteine plus threonine derived from the revertant strain produced 36 g/liter of L-lysine (as hydrochloride salt) when it was cultured in medium containing 100 g/liter of glucose for 72 hr (36% yield). The amount of lysine production by this mutant was greater than those by the resistant mutants derived directly from the original wild strain. The frequency of the appea...