scholarly journals Studies on the mitochondrially bound form of rat brain creatine kinase

1978 ◽  
Vol 170 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Robert F. G. Booth ◽  
John B. Clark
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Mitochondrial Protein ◽  
Mitochondrial Fraction ◽  
Brain Mitochondria ◽  
Adult Brain ◽  
Reverse Direction ◽  
Total Creatine ◽  
Brain Creatine Kinase ◽  
Brain Energy

1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14–15 days after birth, the activity remains less than one-third that of the adult activity (207±6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9±2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100μm) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50μm) or oligomycin (1.5μg/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent Km and Vmax. values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The Vmax. in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent Km for creatine of 1.67mm and for MgATP2−of 0.1mm, and in the reverse direction Vmax. is 489nmol/min per mg of mitochondrial protein, with an apparent Km for phosphocreatine of 0.4mm and for MgADP−of 27μm. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.

scholarly journals Age-Related Changes in Swine Brain Creatine Kinase-Catalyzed 31P Exchange Measured In vivo Using 31P NMR Magnetization Transfer

1994 ◽  
Vol 14 (6) ◽  
pp. 1070-1077 ◽  
Author(s):  
Ronald J. T. Corbett ◽  
Abbot R. Laptook
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Energy Demand ◽  
Creatine Kinase Activity ◽  
Single Frequency ◽  
Forward Rate ◽  
Miniature Swine ◽  
Age Related ◽  
Brain Creatine Kinase

31P exchange rates through the creatine kinase-catalyzed interconversion of phosphocreatine and γ-ATP were measured in a total of 27 miniature swine ranging in age from 5 days preterm to 5 weeks old. A steep increase in the forward rate constant for 31P exchange from phosphocreatine (PCr) to γ-ATP was observed between 2 days preterm and 3 days postterm, with a more gradual increase for older ages. In contrast, the [PCr]/[NTP] ratio measured by in vivo 31P nuclear magnetic resonance (NMR) remained constant throughout this age interval and close to unity. Forward and reverse rate constants and the rate of flux for 31P exchange were equal to each other for both preterm and 5-week-old animals, suggesting that the creatine kinase reaction is near-equilibrium for this span of age. Multifrequency steady-state saturation of Pi and PCr compared to single-frequency saturation of PCr produced the same extent of saturation transfer to γ-ATP, and the saturation of Pi alone had no effect on the γ-ATP 31P NMR signal. These results suggest that even for immature swine brain, creatine kinase activity should be adequate to buffer against changes in [ATP] when there is a mismatch between energy supply and energy demand, during conditions such as ischemia or hypoxia. The results from the present study indicate the unlikelihood that previously reported discrepancies between forward and reverse 32P flux rates in rat brain (Shoubridge et al., FEBS Lett 140:288–292, 1982) were due to neglect of γ-ATP to Pi exchange. If the contribution of nonadenosine triphosphate to the in vivo rat brain 31P NMR signal is accounted for in the calculation of reverse flux and a literature value for rat brain [PCr] is used in the calculation of forward flux, then forward and reverse flux rates are equal.

Rat Brain Creatine Kinase Messenger RNA Levels Are High in Primary Cultures of Brain Astrocytes and Oligodendrocytes and Low in Neurons

1992 ◽  
Vol 59 (5) ◽  
pp. 1925-1932 ◽  
Author(s):  
George R. Molloy ◽  
Charlie D. Wilson ◽  
Pamela Benfield ◽  
Jean deVellis ◽  
Shalini Kumar
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Messenger Rna ◽  
Primary Cultures ◽  
Brain Creatine Kinase ◽  
Rna Levels

scholarly journals Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

1990 ◽  
Vol 10 (12) ◽  
pp. 6533-6543 ◽  
Author(s):  
G M Hobson ◽  
G R Molloy ◽  
P A Benfield
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Hela Cells ◽  
Functional Organization ◽  
Regulatory Elements ◽  
Primer Extension ◽  
Tata Box ◽  
Kinase Gene ◽  
Brain Creatine Kinase

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

scholarly journals Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development

1975 ◽  
Vol 152 (3) ◽  
pp. 477-484 ◽  
Author(s):  
G L Reijnierse ◽  
H Veldstra ◽  
C J Van der Ber
Keyword(s):  
Subcellular Localization ◽  
Rat Brain ◽  
Short Chain Fatty Acids ◽  
Acid Synthesis ◽  
Mitochondrial Fraction ◽  
Rate Of Change ◽  
Adult Brain ◽  
Short Chain ◽  
Acetyl Coa

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.

Expression of the rat brain creatine kinase gene in C6 glioma cells

1993 ◽  
Vol 35 (1) ◽  
pp. 92-102 ◽  
Author(s):  
C. D. Wilson ◽  
B. Parameswaran ◽  
G. R. Molloy
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Kinase Gene ◽  
Brain Creatine Kinase

Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6533-6543
Author(s):  
G M Hobson ◽  
G R Molloy ◽  
P A Benfield
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Hela Cells ◽  
Functional Organization ◽  
Regulatory Elements ◽  
Primer Extension ◽  
Tata Box ◽  
Kinase Gene ◽  
Brain Creatine Kinase

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

Isolation of four rat creatine kinase genes and identification of multiple potential promoter sequences within the rat brain creatine kinase promoter region

Gene ◽  
1988 ◽  
Vol 63 (2) ◽  
pp. 227-243 ◽  
Author(s):  
Pamela A. Benfield ◽  
David Graf ◽  
Peter N. Korolkoff ◽  
Grace Hobson ◽  
Mark L. Pearson
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Promoter Region ◽  
Promoter Sequences ◽  
Potential Promoter ◽  
Brain Creatine Kinase

scholarly journals Synthesis of N-acetyl-l-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport

1979 ◽  
Vol 184 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Tarun B. Patel ◽  
John B. Clark
Keyword(s):  
Rat Brain ◽  
Respiration Rate ◽  
Mitochondrial Protein ◽  
Brain Mitochondria ◽  
Additional Mechanism ◽  
Adult Rat ◽  
Rat Brain Mitochondria ◽  
Adult Rat Brain ◽  
Different Ages ◽  
Carbon Transport

1. The synthesis and efflux of N-acetyl-l-aspartate from brain mitochondria of rats of different ages has been studied. 2. Brain mitochondrial State 3 (+ADP) respiration rate, using 10mm-glutamate and 2.5mm-malate as substrates, increases during the suckling period and reaches approx. 50% of the adult value at 17 days after birth [adult State 3 respiration rate=160±7ng-atoms of O/min per mg of mitochondrial protein(mean±s.d.; n=3)]. 3. The influence of 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate on aspartate efflux from brain mitochondira from rats of different ages oxidizing glutamate and malate was studied. In all cases the aspartate efflux in State 3 was greater than in State 4, but, whereas the aspartate efflux in State 3 increased as the animals developed, that of State 4 showed only a small increase. However, the rate of aspartate efflux in the presence of pyruvate or 3-hydroxybutyrate as well as glutamate and malate was approx. 60–65% of that in the presence of glutamate and malate alone. 4. An inverse relationship between aspartate efflux and N-acetylaspartate efflux was observed with adult rat brain mitochondria oxidizing 10mm-glutamate and 2.5mm-malate in the presence of various pyruvate concentrations (0–5mm). 5. N-Acetylaspartate efflux by brain mitochondria of rats of different ages was studied in States 3 and 4, utilizing 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate as acetyl-CoA sources. A similar pattern of increase during development was seen in State 3 for N-acetylaspartate efflux as for aspartate efflux (see point 3 above). Also only very small increases in N-acetylaspartate efflux occurred during development in State 4.6. Rat brain mitochondria in the presence of iso-osmotic N-acetylaspartate showed some swelling which was markedly increased in the presence of malate. 7. It is concluded that N-acetylaspartate may be synthesized and exported from both neonatal and adult rat brain mitochondria. It is proposed that the N-acetylaspartate is transported by the dicarboxylic acid translocase and may be an additional mechanism for mitochondrial/cytosolic carbon transport to that of citrate.

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