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Some basic questions regarding transport of a metabolite across the inner mitochondrial, membrane
Biookmnicial aapeotsof kidney fnnotfon
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utilizing L-U-[14C]-malateloaded mitochondria. The rates of efflux of malate in the for this transpresence of Pi were measured both in the rat and the rabbit. The V porter (1.6 mU/mg mitochondrialprotein at 5*C) was found to be lo-%d lower in the rabbit than the rat. Metabolic acidosis had no effect on the kinetics of this transporter in either species. In conclusion,the low rate of ammoniagenesisin the rabbit with metabolic acidosis could be due to the low flux through the dicarboxylate transporter.
SOME BASIC FESTOONS RE~RDING T~SPORT OF A ~TABOLITE ACROSS THE INNER MITOCHONDRIALMEMBRANE 2. Kovacevic,M. Pavolvic and K. Bajin Dept. of Biochem, Medical Faculty, Novi Sad, Yugoslavia Despite its importance,the problem of glutamine transport into kidney mitochondria is essentiallystill unresolved. The existence of a specific glutamine carrier is supportedmain1 by the followingobservations: mersalyl decreases %+C glutaminepermeable space. (1) Phosphate-dependent glutaminase (PDG) shows the phenomenonof latency. It (2) is inhibitedby mersalyl in intact mitochondriaand this inhibitionis removed by a detergent. The mitochondriaswell in an iso-osmoticsolution of glutamine. (3) These observationswere questionedon the followinggrounds: It is possible that mersalyl primarily inhibits PDG which might be essential (1) for the transport of glutamine. The removal of the mersalyl inhibitionof PDG by detergentmight be the result (2) of unmasking of additionalSH groups rather than of the removal of a permeability brrier. It has never been verified if the uptake of 14C glutamineby kidney mitochondria (3) representstransport or a binding process. The present work, which will be reported,deals with the above questions and gives a new insight into the problem of glutamineuptake by kidney mitochondria. By increasing hypotonicityofthe incubationmedium it was found that there is no decrease of the inhibitoryeffect of mersalyl which throws doubt that glutamine transport is primarily inhibited. Also, by using different thiol-blockingagents (mersalyl,NEM, pCMB and DTNB) we were able to show that there is a correlationbetween theinhibitionof PDG activity in intact and lysed mitochondria. These and other findings suggest that the present concept of glutaminetransport should be re-evaluated.
EFFECT OF pH ON METABOLISMOF THE GLUTAMINE (GLN) CARBON SKELETON BY RENAL CORTICAL MITOCHONDRIA A.S. Kunin and R.L. Tannen Burlington,Vermont and Ann Arbor, Michigan, USA. The effect of altered acid-basehomeostasison the intramitochondrial metabolism of the gln carbon skeleton was assessed initiallyby determining 14COg production from U-14C-Gln. The response to pH was highly de endent on the gln concentrationemployed, and interpretationwas also complicatedby 1!CO2 productionwith 1,4 1% succinateas the sole substrate. Therefore a more precise analysis was performed by measuring the metabolitesof unlabelledgln. aKG metabolizedwas calculatedas the difference between oKG production (glutamatedeaminationplus transamination)and uKG accumulation in the medium. Under all experimentalconditionsaccumulationin the medium of malate plus aspartate,the major end products of metabolizedaKG, was altered similarly to the calculatedchange in uKG metabolism. Recycling of aKG through the TCA cycle was minimal, since the calculatedrate of metabolismwas very similar in the presence of fluoroacetate. Mitochondriafrom rats with chronic acidosis were compared to pair fed controls using 1.0 or 5.0 mM gln. aKG production and intramitochondrialconcentrationincreased markedly, but aKG metabolismwas unchanged. When normal mitochondriawere incubated at pH 7.0, 7.4 and 7.7, as with 14C02 production,the response was dependent on the concentrationof gln (0.5, 1.0 or 5.0 mM). Analysis of the data indicated that pH simultaneouslyaffected both the production of aKG and its conversionto succinate. A high pH stimulatesproduction but inhibits subsequentmetabolism of aKG, while an acid
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utilizing L-U-[14C]-malateloaded mitochondria. The rates of efflux of malate in the for this transpresence of Pi were measured both in the rat and the rabbit. The V porter (1.6 mU/mg mitochondrialprotein at 5*C) was found to be lo-%d lower in the rabbit than the rat. Metabolic acidosis had no effect on the kinetics of this transporter in either species. In conclusion,the low rate of ammoniagenesisin the rabbit with metabolic acidosis could be due to the low flux through the dicarboxylate transporter.
SOME BASIC FESTOONS RE~RDING T~SPORT OF A ~TABOLITE ACROSS THE INNER MITOCHONDRIALMEMBRANE 2. Kovacevic,M. Pavolvic and K. Bajin Dept. of Biochem, Medical Faculty, Novi Sad, Yugoslavia Despite its importance,the problem of glutamine transport into kidney mitochondria is essentiallystill unresolved. The existence of a specific glutamine carrier is supportedmain1 by the followingobservations: mersalyl decreases %+C glutaminepermeable space. (1) Phosphate-dependent glutaminase (PDG) shows the phenomenonof latency. It (2) is inhibitedby mersalyl in intact mitochondriaand this inhibitionis removed by a detergent. The mitochondriaswell in an iso-osmoticsolution of glutamine. (3) These observationswere questionedon the followinggrounds: It is possible that mersalyl primarily inhibits PDG which might be essential (1) for the transport of glutamine. The removal of the mersalyl inhibitionof PDG by detergentmight be the result (2) of unmasking of additionalSH groups rather than of the removal of a permeability brrier. It has never been verified if the uptake of 14C glutamineby kidney mitochondria (3) representstransport or a binding process. The present work, which will be reported,deals with the above questions and gives a new insight into the problem of glutamineuptake by kidney mitochondria. By increasing hypotonicityofthe incubationmedium it was found that there is no decrease of the inhibitoryeffect of mersalyl which throws doubt that glutamine transport is primarily inhibited. Also, by using different thiol-blockingagents (mersalyl,NEM, pCMB and DTNB) we were able to show that there is a correlationbetween theinhibitionof PDG activity in intact and lysed mitochondria. These and other findings suggest that the present concept of glutaminetransport should be re-evaluated.
EFFECT OF pH ON METABOLISMOF THE GLUTAMINE (GLN) CARBON SKELETON BY RENAL CORTICAL MITOCHONDRIA A.S. Kunin and R.L. Tannen Burlington,Vermont and Ann Arbor, Michigan, USA. The effect of altered acid-basehomeostasison the intramitochondrial metabolism of the gln carbon skeleton was assessed initiallyby determining 14COg production from U-14C-Gln. The response to pH was highly de endent on the gln concentrationemployed, and interpretationwas also complicatedby 1!CO2 productionwith 1,4 1% succinateas the sole substrate. Therefore a more precise analysis was performed by measuring the metabolitesof unlabelledgln. aKG metabolizedwas calculatedas the difference between oKG production (glutamatedeaminationplus transamination)and uKG accumulation in the medium. Under all experimentalconditionsaccumulationin the medium of malate plus aspartate,the major end products of metabolizedaKG, was altered similarly to the calculatedchange in uKG metabolism. Recycling of aKG through the TCA cycle was minimal, since the calculatedrate of metabolismwas very similar in the presence of fluoroacetate. Mitochondriafrom rats with chronic acidosis were compared to pair fed controls using 1.0 or 5.0 mM gln. aKG production and intramitochondrialconcentrationincreased markedly, but aKG metabolismwas unchanged. When normal mitochondriawere incubated at pH 7.0, 7.4 and 7.7, as with 14C02 production,the response was dependent on the concentrationof gln (0.5, 1.0 or 5.0 mM). Analysis of the data indicated that pH simultaneouslyaffected both the production of aKG and its conversionto succinate. A high pH stimulatesproduction but inhibits subsequentmetabolism of aKG, while an acid