Phosphate transport, mitochondrial

Figure 12-10. Transporter systems in the inner mitochondrial membrane. , phosphate transporter ...

In mitochondrial research the phosphate-transporting protein from rat liver mitochondria has been labeled with ° Hg-mersalyl At For protein labeling with astatine (alpha emitter) the following procedures may be u reaction of the protein with p-astatobenzoic acid < ndensation reaction with peptide bond and protein acetylation While labeling by the above procedures seems to be sufficiently stable a remarkable instability of the At-label was obserwd when astatinated protein was prepared electrophoretically 202) jjjg results of these authors indicate that the tyrosine-astatine bond is unstable. The conclusion of Vau an et al. that astatinated proteins lore as much as 50% of their biological activity and, in addition, are extremely toxic, is very serious. 

The pyruvate, glutamate and phosphate transporters catalyze net uptake and release of their substrates with stoicheiometric amounts of protons [6]. Early evidence for the electroneutrality of the process was the good inverse correlation between the H gradient across the mitochondrial membrane and the gradients of these permeant anions, especially at equilibrium and at low metabolite concentrations [96,97]. At equilibrium the rate of inward transport should equal the rate of efflux and the distribution of permeant anion should be proportional to the A pH since ... 

A number of workers have been able to isolate a protein, covalently linked to radioactive Af-ethylmaleimide, identified as the mitochondrial phosphate transporter [122,193-196]. The isolation and identification of the transporter was based for the most part on the maleimide and mersalyl reactivity of the protein. The molecular weight of the protein isolated from different sources varies from 27000 to 34000. Because of the covalent linkage to the irreversible inhibitor, reconstitution of transport was not feasible. 

In addition to powering ATP synthesis, the proton-motive force across the inner mitochondrial membrane also powers the exchange of ATP formed by oxidative phosphorylation inside the mitochondrion for ADP and Pj in the cytosol. This exchange, which is required for oxidative phosphorylation to continue, is mediated by two proteins in the inner membrane a phosphate transporter (HP04 /OH antiporter) and an ATP/ADP antiporter (Figure 8-28). 

The phosphate transporter catalyzes the import of one HP04 coupled to the export of one OH . Likewise, the ATP/ADP antiporter allows one molecule of ADP to enter only if one molecule of ATP exits simultaneously. The ATP/ADP antiporter, a dimer of two 30,000-Da subunits, makes up 10-15 percent of the protein in the inner membrane, so it is one of the more abundant mitochondrial proteins. Functioning of the two antiporters together produces an Influx of one ADP and one Pj and efflux of one ATP together with one OH . Each OH transported outward combines with a proton, translocated during electron transport to the Intermembrane space, to form H2O. This drives the overall reaction in the direction of ATP export and ADP and Pj Import. 

Ascofuranone (9), an isoprenoid prenylphenol antibiotic, derived from the fungus Ascochyta visiae, specifically inhibits mitochondrial glycerol-3-phosphate (G-3-P)-dependent electron transport in T. b. brucei [44]. Ascofuranone strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial oxygen consumption of T. b. brucei bloodstream form... 

In studies performed by Wohlrab [108] and by Hofmann and Kadenbach [109] a mitochondrial iimer membrane protein with an M of 31000-32000 with either Af-[ H]ethylmaleimide [108] or [ Hg]mersalyl [109] bound to it was isolated, which was claimed to be the phosphate translocator. Proof of its identity, however, can only be obtained if the protein is able to mediate phosphate transport in a reconstituted system. 

Fig. 3. Primary carbon metabolism in a photosynthetic C3 leaf. An abbreviated depiction of foliar C02 uptake, chloroplastic light-reactions, chloroplastic carbon fixation (Calvin cycle), chloroplastic starch synthesis, cytosolic sucrose synthesis, cytosolic glycolysis, mitochondrial citric acid cycle, and mitochondrial electron transport. The photorespiration cycle spans reactions localized in the chloroplast, the peroxisome, and the mitochondria. Stacked green ovals (chloroplast) represent thylakoid membranes. Dashed arrows near figure top represent the C02 diffusion path from the atmosphere (Ca), into the leaf intercellular airspace (Ci), and into the stroma of the chloroplast (Cc).SoHd black arrows represent biochemical reactions. Enzyme names and some substrates and biochemical steps have been omitted for simplicity. The dotted line in the mitochondria represents the electron transport pathway. Energy equivalent intermediates (e.g., ADP, UTP, inorganic phosphate Pi) and reducing equivalents (e.g., NADPH, FADH2, NADH) are labeled in red. Membrane transporters Aqp (CO2 conducting aquaporins) and TPT (triose phosphate transporter) are labeled in italics. Mitochondrial irmer-membrane electron transport and proton transport proteins are labeled in small case italics.

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

Figure 12-14. The creatine phosphate shuttle of heart and skeletal muscle. The shuttle allows rapid transport of high-energy phosphate from the mitochondrial matrix into the cytosol. CKg, creatine kinase concerned with large requirements for ATP, eg, muscular contraction CIC, creatine kinase for maintaining equilibrium between creatine and creatine phosphate and ATP/ADP CKg, creatine kinase coupling glycolysis to creatine phosphate synthesis CK, , mitochondrial creatine kinase mediating creatine phosphate production from ATP formed in oxidative phosphorylation P, pore protein in outer mitochondrial membrane.

The P/O ratio is the number of ATPs made for each O atom consumed by mitochondrial respiration. The P stands for high-energy phosphate equivalents, and the O actually stands for the number of I 02 s that are consumed by the electron transport chain. The full reduction of 02 to 2 H20 takes 4 electrons. Therefore, 2 electrons reduce of an 02. The oxidation of NADH to NAD and the oxidation of FADH2 to FAD are both 2-electron oxidations. O can be read as the transfer of 2 electrons. It s not quite as obscure as it sounds.2... 

The final reactions to be considered in the metabolism of ethanol in the liver are those involved in reoxidation of cytosolic NADH and in the reduction of NADP. The latter is achieved by the pentose phosphate pathway which has a high capacity in the liver (Chapter 6). The cytosolic NADH is reoxidised mainly by the mitochondrial electron transfer system, which means that substrate shuttles must be used to transport the hydrogen atoms into the mitochondria. The malate/aspartate is the main shuttle involved. Under some conditions, the rate of transfer of hydrogen atoms by the shuttle is less than the rate of NADH generation so that the redox state in the cytosolic compartment of the liver becomes highly reduced and the concentration of NAD severely decreased. This limits the rate of ethanol oxidation by alcohol dehydrogenase. 

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