Absence Of Exogenous ATP Research Articles

Role of substrates in Sr 2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr 2+-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr 2+ and did not take up any of the released K +. Addition of substances decreasing the mitochondrial NADH NAD + ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K + and for complete uptake of Sr 2+ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K + and Sr 2+ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.

Avian mitochondrial glutamine metabolism.

Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.

Synthesis of Fatty Acids in Outer and Inner Membranes of Mitochondria

Fatty acids can be synthesized in mitochondrial membranes of rat liver either by elongation or by synthesis de novo. The outer membranes incorporate greater amounts of substrate into fatty acids than do the inner membranes. Outer membranes synthesize 6- and 8-carbon fatty acids completely de novo, while smaller amounts of 10-carbon acids are produced by the mechanism de novo. The reactions de novo utilize acetyl coenzyme A more effectively than malonyl coenzyme A, and synthesis de novo occurs in the absence of exogenous ATP. Elongation, which produces fatty acids 14 carbon atoms long and longer, depends on added ATP, and acetyl-CoA and malonyl-CoA are incorporated about equally. Synthesis de novo of shorter chain fatty acids by outer membranes continues in the presence of ATP and the concomitant elongation reactions. Inner membranes synthesize only longer fatty acids by elongation. If synthesis de novo of fatty acids occurs in the inner membranes, these acids might be removed by oxidation. The ATP concentrations in these experiments were compared with the levels of ATP produced by actively respiring liver mitochondria. During metabolically adverse conditions within the cell, the capacity for manufacturing fatty acids by elongation may fluctuate with the ATP levels whereas synthesis de novo continues.