scholarly journals Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.

1987 ◽  
Vol 105 (6) ◽  
pp. 2631-2639 ◽  
Author(s):  
E S Sztul ◽  
J P Hendrick ◽  
J P Kraus ◽  
D Wall ◽  
F Kalousek ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Liver Mitochondria ◽  
Essential Elements ◽  
Ornithine Transcarbamylase ◽  
Proteolytic Processing ◽  
Leader Peptide ◽  
Rat Liver Mitochondria

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

Import of the glucocorticoid receptor into rat liver mitochondria in vivo and in vitro

1993 ◽  
Vol 46 (3) ◽  
pp. 401-413 ◽  
Author(s):  
C. Demonacos ◽  
N.C. Tsawdaroglou ◽  
R. Djordjevic-Markovic ◽  
M. Papalopoulou ◽  
V. Galanopoulos ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria

The Effect of Insulin on Mitochondrial Particles

1967 ◽  
Vol 25 ◽  
pp. 160-161 ◽  
Author(s):  
Burton B. Silver ◽  
James C. Hall
Keyword(s):  
Bovine Serum Albumin ◽  
Structural Changes ◽  
Normal Values ◽  
Liver Mitochondria ◽  
Synergistic Action ◽  
Diabetic Rat ◽  
Rat Liver Mitochondria ◽  
Structural Studies

Correlative biochemical and structural studies have shown that insulin and Mg++ may act to alter the configuration and also enhance the efficiency of coupled phosphorylation in sonicated fragments of diabetic rat liver mitochondria. The diabetic preparations had consistently lowered P:O ratios which returned to normal values with addition of insulin in vivo or in vitro. Optimum coupling and structural changes with insulin required a Mg++ concentration of 5 × 10−5 M. Insulin remained effective diluted to a concentration of 2 × 10−4 I.U. per ml. Glutathione, bovine serum albumin, and Zn++ were ineffective in producing either coupling or structural changes. There seems to be a synergistic action of insulin and Mg++ in restoring P:O ratios in diabetic particles while simultaneously altering the structure toward normal control appearances. Fragments negatively stained with phosphotungstate indicated that the normal particles had well defined cristae with numerous evenly distributed, stalked subunits, 90 Å in diameter.

scholarly journals Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

1977 ◽  
Vol 164 (3) ◽  
pp. 685-691 ◽  
Author(s):  
E Marra ◽  
S Doonan ◽  
C Saccone ◽  
E Quagliariello
Keyword(s):  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Transferase Activity ◽  
Selective Permeability ◽  
The Matrix ◽  
Mitochondrial Aspartate Aminotransferase

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

scholarly journals Role of polyamines in the transport in vitro of the precursor of ornithine transcarbamylase

1991 ◽  
Vol 279 (3) ◽  
pp. 815-820 ◽  
Author(s):  
C González-Bosch ◽  
M J Marcote ◽  
J Hernández-Yago
Keyword(s):  
Rat Liver ◽  
Signal Sequence ◽  
Chimeric Protein ◽  
Liver Mitochondria ◽  
Ornithine Transcarbamylase ◽  
Rat Liver Mitochondria ◽  
Surface Transport ◽  
Binding To Mitochondria

Polyamines induce the transport in vitro of the rat liver precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria. The accumulation of this precursor at the level of binding to the mitochondrial surface has allowed us to establish that polyamines are involved in the interaction of the precursor with the mitochondrial surface. Transport of a chimeric protein having the signal sequence of pOTC fused to a fragment of the cytosolic protein human arginosuccinate lyase was also induced by polyamines. The sensitivity of the pOTC synthesized in vitro and of the chimeric protein to proteinases decreases in the presence of polyamines. This result suggests that polyamines may play a role in modulating the folding of precursors to favour their binding to mitochondria.

scholarly journals Specificity of human rhinovirus 2Apro is determined by combined spatial properties of four cleavage site residues

2013 ◽  
Vol 94 (7) ◽  
pp. 1535-1546 ◽  
Author(s):  
David Neubauer ◽  
Martina Aumayr ◽  
Irene Gösler ◽  
Tim Skern
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Cleavage Site ◽  
Antiviral Agents ◽  
Genetic Group ◽  
Human Rhinovirus ◽  
C Terminus ◽  
Group B

The 2A proteinase (2Apro) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2Apro substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2Apro of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2Apro (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2′, HRV1A and HRV2 2Apro cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2Apro were substituted (e.g. at P2, P1 and P2′), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2′ were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2Apro revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

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