scholarly journals Protein degradation in rat liver during post-natal development

1980 ◽  
Vol 192 (1) ◽  
pp. 321-330 ◽  
Author(s):  
S M Russell ◽  
R J Burgess ◽  
R J Mayer
Keyword(s):  
Protein Degradation ◽  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Adult Rats ◽  
Adult Rat ◽  
Mitochondrial Inner Membrane ◽  
Degradation Rates ◽  
A Subunit

Protein degradation rates for liver subcellular and submitochondrial fractions from neonatal (8-day), weanling (25-day) and adult rats were estimated by the double-isotope method with NaH14CO3 and [3H] arginine as the radiolabelled precursors [Dice, Walker, Byrne & Cardiel (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097]. Decreased protein degradation rates were found during post-natal development for homogenate, nuclear, mitochondrial, lysosomal and microsomal proteins. A decrease in degradation rates for the immunoisolated subunits of monoamine oxidase and pyruvate dehydrogenase was also observed in neonatal and weanling rats respectively. The results suggest coordinate degradation of the subunits of the multi-subunit enzyme pyruvate dehydrogenase. Pyruvate dehydrogenase has a faster rate of degradation in adult rat liver than does cytochrome oxidase. Data analysis suggests heterogeneity of protein degradation rates in the mitochondrial outer membrane and intermembrane space fractions at each developmental stage but not in the mitochondrial inner membrane or matrix fractions. Results obtained for protein degradation rates in adult rat liver by the method of Burgess, Walker & Mayer [(1978) Biochem. J. 176, 919-926] in general confirmed the results obtained for the adult rat liver by the above method. No evidence of a subunit-size relationship for protein degradation was found for proteins in any subcellular or submitochondrial fraction.

Postnatal expression of the canalicular bile acid transport system of rat liver

1991 ◽  
Vol 260 (5) ◽  
pp. G743-G751 ◽  
Author(s):  
D. A. Novak ◽  
C. J. Sippel ◽  
M. Ananthanarayanan ◽  
F. J. Suchy
Keyword(s):  
Bile Acid ◽  
Rat Liver ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Precipitation Method ◽  
Disulfonic Acid ◽  
Acid Transport ◽  
Adult Rats ◽  
Bile Acid Transport ◽  
Adult Rat

Canalicular plasma membrane (CPM) vesicles prepared by a Ca2+ precipitation method from developing (7 and 14 days old) and adult rat liver were used to directly examine the postnatal ontogenesis of taurocholate (TC) transport. The initial rate of 50 microM TC uptake by vesicles derived from 14-day-old and adult but not 7-day-old animals was markedly inhibited by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). DIDS-sensitive TC uptake was 21.6 +/- 5.6 (SE) at 14 days compared with 58.1 +/- 8.1 pmol.mg protein-1.5 s-1 in adults (P less than or equal to 0.01). Kinetic studies were performed by preloading these predominantly "right-side out" vesicles with TC (25-800 microM) and measuring the initial rate (5 s) of efflux into bile salt-free medium. Computer analysis of the DIDS-sensitive portion of efflux revealed saturable kinetics with a similar Vmax (2.72 +/- 0.36 vs. 1.97 +/- 0.17 nmol.mg protein-1.min-1; P = NS) but a threefold higher Km (0.35 +/- 0.09 vs. 0.11 +/- 0.02 mM; P less than or equal to 0.05) in 14 day vs. adult CPM vesicles. In contrast, efflux from 7 day CPM vesicles increased linearly with increasing concentrations of TC and was not inhibited by DIDS. Immunoblots of canalicular membranes, probed with an antibody against the 100-kDa bile acid transport protein, showed that the amount of immunoreactive carrier protein in the membranes of 14-day-old and adult rats was similar but was only 37% of the adult level at 7 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors

1992 ◽  
Vol 282 (3) ◽  
pp. 909-914 ◽  
Author(s):  
K Kashfi ◽  
G A Cook
Keyword(s):  
Outer Membrane ◽  
Adenylate Kinase ◽  
Citrate Synthase ◽  
Physiological State ◽  
Carnitine Palmitoyltransferase ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
No Release

Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states.

Hepatic stimulator substance: physicochemical characteristics and specificity

1982 ◽  
Vol 242 (3) ◽  
pp. G281-G288 ◽  
Author(s):  
D. R. LaBrecque ◽  
N. R. Bachur
Keyword(s):  
Rat Liver ◽  
Nuclear Dna ◽  
Tritiated Thymidine ◽  
Fold Increase ◽  
Physicochemical Characteristics ◽  
Adult Rats ◽  
Hepatic Stimulator Substance ◽  
Adult Rat ◽  
Heat Stable ◽  
Organ Specific

This laboratory has reported previously that a cytoplasmic extract of weanling or regenerating adult rat liver (but not normal rat liver) will produce a 2.5-fold increase in the incorporation of tritiated thymidine ([3H]dThd) into liver DNA of a 34%-hepatectomized test animal. (J. Physiol. London 248: 273-284, 1975). The present study showed that hepatic stimulator substance (HSS) will stimulate DNA synthesis in normal adult rats and CF1 mice as well. The increased incorporation of [3H]dThd into DNA produced in the normal, nonhepatectomized adult rat was comparable with that induced by a 34% hepatectomy. Autoradiographic studies revealed that the [3H]dThd was incorporated into nuclear DNA and that the stimulation occurred almost exclusively in parenchymal cells. HSS was shown to be heat stable (100 degrees C for 15 min) and was precipitated but not inactivated by alcohol. Ultrafiltration and dialysis studies suggested a molecular weight slightly greater than 10,000. HSS proved to be organ specific, stimulating the liver but not the kidney, bone marrow, or spleen. HSS was found to contain no insulin, glucagon, epidermal growth factor, or peptides of the nonsuppressible insulinlike/multiplication-stimulating activity (somatomedin) group.

scholarly journals Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria

1985 ◽  
Vol 231 (3) ◽  
pp. 581-595 ◽  
Author(s):  
J G McCormack
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Pyruvate Dehydrogenase Kinase ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Mammalian Tissues ◽  
14Co2 Production

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent ‘State 3.5’ respiration condition. Ca2+ had no effect on NAD(P)H formation induced by β-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.

scholarly journals Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria

2000 ◽  
Vol 150 (6) ◽  
pp. 1271-1282 ◽  
Author(s):  
Alison J. Davis ◽  
Naresh B. Sepuri ◽  
Jason Holder ◽  
Arthur E. Johnson ◽  
Robert E. Jensen
Keyword(s):  
Protein Complexes ◽  
Intermediate Stage ◽  
Terminal Segment ◽  
Mitochondrial Outer Membrane ◽  
Cross Linking ◽  
Intermembrane Space ◽  
Cross Link ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

scholarly journals Effect of hypophysectomy on the rates of protein synthesis and degradation in rat liver

1979 ◽  
Vol 178 (3) ◽  
pp. 725-731 ◽  
Author(s):  
R D Conde
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Rat Liver ◽  
Protein Metabolism ◽  
Plasma Proteins ◽  
Degradation Rates ◽  
Hypophysectomized Rats ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

The effect of hypophysectomy on the protein metabolism of the liver in vivo was studied. Fractional rates of protein synthesis and degradation were determined in the livers of normal and hypophysectomized rats. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was estimated either as the balance between synthesis and accumulation of stable liver proteins or from the disappearance of radioactivity from the proteins previously labelled by the injection of NaH14CO3. The results indicate that: (1) hypophysectomy diminishes the capacity of the liver to synthesize proteins in vivo, mainly of those that are exported as plasma proteins; (2) livers of both normal and hypophysectomized rats show identical protein-degradation rates, whereas plasma proteins are degraded slowly after hypophysectomy.

scholarly journals Biogenesis of Mitochondrial Metabolite Carriers

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1008 ◽  
Author(s):  
Patrick Horten ◽  
Lilia Colina-Tenorio ◽  
Heike Rampelt
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Chaperone Function ◽  
Mitochondrial Inner Membrane ◽  
Metabolic Reactions ◽  
Almost All ◽  
Related Proteins ◽  
Shed Light

Metabolite carriers of the mitochondrial inner membrane are crucial for cellular physiology since mitochondria contribute essential metabolic reactions and synthesize the majority of the cellular ATP. Like almost all mitochondrial proteins, carriers have to be imported into mitochondria from the cytosol. Carrier precursors utilize a specialized translocation pathway dedicated to the biogenesis of carriers and related proteins, the carrier translocase of the inner membrane (TIM22) pathway. After recognition and import through the mitochondrial outer membrane via the translocase of the outer membrane (TOM) complex, carrier precursors are ushered through the intermembrane space by hexameric TIM chaperones and ultimately integrated into the inner membrane by the TIM22 carrier translocase. Recent advances have shed light on the mechanisms of TOM translocase and TIM chaperone function, uncovered an unexpected versatility of the machineries, and revealed novel components and functional crosstalk of the human TIM22 translocase.

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