An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane

Kyriazi, Harold T.; Basford, R. E.

doi:10.1016/0003-9861(86)90423-6

Cited by 12 publications

(12 citation statements)

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“…In addition, it has been shown that one ofthe actions ofinsulin is to increase the binding ofhexokinase to mitochondria in heart, skeletal muscle, and brain (13)(14)(15)(16) and that binding decreases the ability of glucose 6-phosphate to inhibit the enzyme ( 17,18). Because the association of hexokinase with mitochondria could alter the apparent affinity constant (Km) for either glucose or 2-deoxyglucose, it is necessary to determine if differences do indeed exist in the affinity constants for glucose and 2-deoxyglucose for hexokinase that is bound to mitochondrial particles.…”

Section: Introductionmentioning

confidence: 99%

Compartmentation of hexokinase in rat heart. A critical factor for tracer kinetic analysis of myocardial glucose metabolism.

Russell¹,

Mrus

Mommessin

et al. 1992

J. Clin. Invest.

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Radiolabeled analogues of 2-deoxyglucose are widely used to trace glucose metabolism in cell cultures, whole organs, and intact animals, although kinetic differences in transport and phosphorylation between these compounds and glucose exist. The present studies were undertaken to determine the effects of insulin stimulation on the phosphorylation of 2-deoxyglucose compared to glucose in the intact, saline-perfused working rat heart. Rates of glucose utilization determined from tritiated glucose differed from rates estimated from the accumulation of I 'CI2-deoxyglucose in a nonconstant manner when comparing rates in the absence or presence of physiologic levels of insulin (13 pU/ml). The fraction of monophosphorylated hexoses that was accounted for by ['4C]2-deoxyglucose 6-phosphate was dramatically decreased in hearts perfused in the presence of insulin. Additionally, hexokinase activity associated with the mitochondrial fraction of tissue extracts was increased in hearts stimulated by insulin. While this redistribution of hexokinase to the mitochondria did not affect the apparent affinity constant for glucose, hexokinase bound to mitochondria exhibited an 8.5-fold decrease in the affinity for 2-deoxyglucose when compared with hexokinase present in the cytosolic fraction. The findings are consistent with an insulin-mediated preferential uptake and phosphorylation of glucose compared to deoxyglucose. The results also imply that the redistribution of hexokinase and the differential effect of insulin on its affinity for tracer and tracee are responsible for changes in the "lumped constant" (i.e., the correction factor used to equate 2-deoxyglucose to glucose uptake). These changes must be taken into account when regional myocardial glucose metabolism is assessed by the 2-deoxyglucose method. (J.

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Section: Introductionmentioning

confidence: 99%

Compartmentation of hexokinase in rat heart. A critical factor for tracer kinetic analysis of myocardial glucose metabolism.

Russell¹,

Mrus

Mommessin

et al. 1992

J. Clin. Invest.

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“…To perform experiments described elsewhere (Kyriazi and Basford 1986), it was necessary to obtain a preparation of Ss structurally and metabolically intact, and relatively free of contamination by "free" mitochondria (mitochondria not contained within a plasma membrane-bounded particle). Several methods of Ss preparation were employed.…”

Section: Resultsmentioning

confidence: 99%

“…Attempts were made to determine if the high levels of Ss AMP were indeed in the intrasynaptosomal mitochondrial matrix, by fractionating the Ss with digitonin in a series of low and increasing ratios of milligrams of digitonin to milligrams of Ss protein for short periods of time (18 or 30 s) prior to centrifugation, to see whether ATP and PCr were released by these treatments to greater extents than was AMP. This would be expected at low milligrams of digitonin/milligrams of Ss protein ratios (if the AMP were within the intrasynaptosomal mitochondrial matrix), because the inner membrane of the intrasynaptosomal mitochondria is not disrupted at these low ratios [as determined by lack of citrate synthase release (Kyriazi and Basford, 1986)]. These experiments failed to demonstrate any differences in the release of ATP, PCr, and AMP.…”

Section: Partsmentioning

confidence: 97%

“…The estimates of the percent contributions (of water and protein) from free mitochondria, CPs, MCPs, and debris to Ss fractions were based on values of either 4.0 (Rafalowska et al, 1980) or 3.23 (Scott and Nicholls, 1980) pl of intraparticle H,O/mg Ss protein. The assumptions made were that the free mitochondria contributed 8% of the total protein in Ss fractions (Kyriazi and Basford, 1986), that the CPs contained a percentage of intraparticle water equal to their percentage of TAN content (from estimates of TANS in CPs derived from calculations like those of Calculation l), and that the remainder of the water was in the free mitochondria and MCPs. With the contributions to the total water content by the free mitochondria and CPs thus fixed, the amount of MCPs was adjusted to give 4.0 or 3.23 pl of total water content.…”

Section: Calculation 2: Estimates Of Intraparticle Protein and Water mentioning

confidence: 99%

“…For sake of calculation, a range of H K activity of 0.001-0.002 U/mg in the CPs is assumed. Table 1 shows that the total HK specific activity of Ss is -0.37, of which about 20-25% (22.5% average) is soluble (Kyriazi and Basford, 1986). Assuming that 35%, 55% or 64% of this soluble HK (22.5% of 0.37 U = 0.083 U) is present in the CPs yields O.O29U, O.O46U, or 0.053U in these particles.…”

Section: Calculation 3: Estimates Of Atp Content In Cytosolic Particlesmentioning

confidence: 99%

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Intractable Unphysiologically Low Adenylate Energy Charge Values in Synaptosome Fractions: An Explanatory Hypothesis Based on the Fraction's Heterogeneity

Kyriazi

Basford

1986

Journal of Neurochemistry

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Synaptosomes prepared and incubated in a variety of ways from rat cerebra exhibited intractable, unphysiologically low adenylate energy charge values (-0.37-0.60), low total adenine nucleotide contents (-8-10 nmol/mg protein), and much higher adenylate kinase apparent Keq values (-3-8) as compared to intact brain tissue (values of -0.90, 25 nmol/mg, and 0.74, respectively). Synaptosomes prepared from mouse, dog, and chicken cerebra had values essentially identical to those from rat. When incubated under oxygen in a physiological salt solution containing glucose, synaptosomes metabolized more glucose to lactic acid than to CO,, and the addition of 100 pM veratridine caused a two-to threefold stimulation of O2 uptake, lactate accumulation, and CO, output. It is known that synaptosome fractions contain a substantial number (at least 30-45% by volume) of cytoplasm-containing particles devoid of mitochondria (henceforth termed "cytosolic particles"), and that -80% of brain hexokinase is bound to the outer mitochondria1 membrane. For the cytosolic particles, lacking oxidative phosphorylation, to maintain their "in vivo" ATP turnover would require about a 19-fold increase in the glycolytic rate, which is not possible due to limiting amounts of hexokinase, and thus these particles are postulated to be responsible for the high level of aerobic lactate accumulation and the intractable Iow energy charge values found in synaptosome fractions. The mitochondria-containing particles are postulated to have a normal energy charge, a submaximal glycolytic rate, and minimal lactate production, on the basis of the capacity of veratridine to stimulate synaptosomal O2 uptake and CO;, and lactate output. Calculations based on this "two populations of particles" hypothesis indicate that for synaptosome fractions in general, (1) the cytosolic particles contain -35-64% of the total adenine nucleotides and maintain an energy charge of -0.12; (2) the cytosolic particles and mitochondria-containing particles have adenylate kinase apparent Keq values of -0.21 -1.66 and 0.74, respectively, revealing that the higher apparent Kcq values of the synaptosome fractions probably are not real departures from equilibrium; and (3) -31-45% of synaptosome fraction protein is contained in debris, which, when taken into account, yields total adenine nucleotide contents in the cytosolic particles and mitochondria-containing particles of -15-24 and -11 -19 nmol/mg of particle protein, respectively. Key Words: Adenine nucleotides-Energy charge-Energy metabolism-Glycolysis-Synaptosomes. Kyriazi H. T. and Basford R. E. Intractable unphysiologically low adenylate energy charge values in synaptosome fractions: An explanatory hypothesis based on the fraction's heterogeneity. J . Neurochem. 47, 512-528 (1986).The s y n a p s e is t h e cardinal feature of neural tissue, and preparations of isolated, "purified" synaptosomes (Ss; detached presynaptic nerve terminals created by the shearing force resulting from the homogenization of neural tissue) have been used widely...

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CTP: Cholinephosphate cytidylyltransferase in human and rat lung: Association in vitro with cytoskeletal actin

Hunt

Normand

Postle

1990

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metaboli

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An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane

Cited by 12 publications

References 68 publications

Compartmentation of hexokinase in rat heart. A critical factor for tracer kinetic analysis of myocardial glucose metabolism.

Compartmentation of hexokinase in rat heart. A critical factor for tracer kinetic analysis of myocardial glucose metabolism.

Intractable Unphysiologically Low Adenylate Energy Charge Values in Synaptosome Fractions: An Explanatory Hypothesis Based on the Fraction's Heterogeneity

CTP: Cholinephosphate cytidylyltransferase in human and rat lung: Association in vitro with cytoskeletal actin

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