Tadao Hashimoto

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus): I. Interaction between cell membranes and virus in the early stage☆

Abstract A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F1F0-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F1F0-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F1F0-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F1 in a manner similar to the inhibitor. The 15K protein binds to the F0 part and holds the inhibitor and the 9K protein on F1F0-ATPase even when one of them is detached from the F1 part.

Levels of purine compounds in a perfusate as a biochemical marker of ischemic injury of cold-preserved liver.

Biochemical markers of ischemic injury of rat liver were studied in an extracorporeal perfusion system. During anoxic perfusion, purine compounds appeared in the perfusate as soon as they were formed in the liver and their recovery in the perfusate balanced the loss of adenine nucleotides from the liver. In contrast, cytosolic aspartate aminotransferase did not appear in the perfusate at slow rates of liver perfusion or during hypothermic perfusion. The production of purine compounds was further investigated in hypothermically preserved liver in connection with the restoration of some metabolic functions of liver. The amount of purine compounds released into the perfusate was found to be closely related to the degrees of damage of the hepatic functions of gluconeogenesis, ureogenesis, and mitochondrial respiration on reperfusion. These results indicate that release of purine compounds into the perfusate is a good marker of ischemic damage.

Interaction with mitochondrial membranes of a synthetic peptide with a sequence common to extra peptides of mitochondrial precursor proteins

A hepta-peptide, Arg-Leu-Leu-Pro-Ser-Leu-Gly, which has a sequence involved in the extra peptides of mitochondrial proteins, was synthesized chemically. The peptide was found to bind specifically to mitochondria, but not to microsomes. The binding was blocked by pretreatment of mitochondria with trypsin but was not affected by the presence of apocytochrome c. The synthetic peptide inhibited the binding to mitochondria of the precursor protein of ATPase inhibitor, which was synthesized in vitro, but did not inhibit that of the precursor of the 9 K stabilizing factor, which has an entirely different extra-peptide sequence. The peptide also did not inhibit the binding of apocytochrome c. These results suggest the existence of a common protein receptor on mitochondrial membranes that facilitates entrance of a group of mitochondrial precursor proteins, including pre-ATPase inhibitor.

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus): II. Effects of pretreatment with a small number of HVJ☆

Abstract Ehrlich ascites tumor cell membranes were completely modified after incubation at 37 °C for 30 min with a small dose of HVJ (about 0.7% of the maximum number of the virus particles that could be adsorbed onto the cells). After this treatment, the cells could adsorb further added HVJ onto their surfaces at 0 °C. But the cell agglutination which was induced by viral adsorption at 0 °C was very weak, and the interaction of the adsorbed virus with the lipid layer of the cell membrane at 37 °C preceding fusion or lysis of the cells was not strong. A discrepancy was observed between acquisition of the modification and liberation of sialic acid (destruction of viral receptors) by viral neuraminidase. The modification proceeded well on incubation at 37 °C but not at lower temperatures. The possibility that the modification is induced by fusion of viral envelopes with cell membranes is discussed.

Primary structure of a regulating factor, 15 kDa protein, of ATP synthase in yeast mitochondria

The amino acid sequence of a factor, 15 kDa protein, which facilitates the formation of, and stabilizes the inactivated complex between mitochondrial ATPase and intrinsic inhibitor was established. The factor was found to be composed of 83 amino acid residues and to have an M r of 9450, though the apparent M r was 15 000 when determiend by SDS—polyacrylamide gel electrophoresis. The factor was characterized as a basic protein with 18 basic and 12 acidic amino acid residues. The amino acid sequence of the factor showed significant homology with those of yeast ATPase inhibitor and the 9 kDa protein which acts in concert with the 15 kDa protein in stabilizing the inactivated ATPase complex.

Novel coulometric detector for high-performance liquid chromatography

Abstract A sensitive coulometric detector was developed for determining electrochemically active compounds in high-performance liquid chromatography. An extremely thin flow layer was formed between a wide glassy carbon electrod and a pore-glass plate, and thus the volume of the detection passageway was sufficiently small. A reference and an auxiliary electrode were placed in an electrolyte chamber on the other side of the pore-glass plate, and thus a potential drip across the extremely thin flow layer on the wide working electrode was completely avoided. The detection limit was 10 fmole for catecholamines in chromatography and 1 fmole in injection to constant flow.

Purification of astroprotein (astrocyte-specific cerebroprotein) by reversed-phase C-1 HPLC.

Abstract: A method for purification of astroprotein (an astrocyte‐specific cerebroprotein) with HPLC is described. A linear gradient from 30 to 70% acetonitrile in 0.1% trifluoroacetic acid (pH 2.2) was applied to the re‐versed‐phase C‐l (particle size 10 μm) column. Cerebroproteins from the crude extract from human glioma were clearly separated by this procedure. Highly purified astroprotein was homogeneous by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and has immunoreactivity to antiserum against astroprotein. Reversed‐phase C‐1 HPLC offers advantages over previously available preparative techniques in the higher purity and better separation time of the products.

Inhibition of the Uncoupler-Induced Mitochondrial ATP-Hydrolysis by the Cooperative Work of the ATPase Inhibitor, 9K Protein and 15K Protein

Yeast mitochondrial F1Fo-ATPase has three regulatory proteins, ATPase inhibitor, 9K protein and 15K protein. Binding of ATPase inhibitor to the enzyme is facilitated and stabilized by cooperative work of 9K protein and 15K protein [Hashimoto, T. et al.(1986) J. Biochem. 99, 251–256]. In the present study, we constructed mutant yeasts lacking in ATPase inhibitor, 9K protein or 15K protein. ATP-synthesizing activities of mitochondria from these mutant yeasts were similar to that of wild-type mitochondria. An uncoupler, CCCP, induced ATP hydrolysis in ATPase inhibitor-deficient mitochondria but not in normal mitochondria. Mitochondria from 9K protein-deficient and 15K protein-deficient cells also exhibited the uncoupler-induced ATP hydrolysis, although their rates were smaller than that of the inhibitor-deficient mitochondria. Submitochondrial particles from the 15K protein-deficient cells exhibited low ATPase activity. However, its ATPase activity gradually increased during the incubation in diluted buffer, while that of wild-type cells remained low. These observations strongly suggest that the ATPase inhibitor acts only to inactivate the ATP-hydrolyzing activity of F1Fo-ATPase when the membrane potential disappeared, and that 15K protein and 9K protein act to reinforce this action of the inhibitor protein.

Interaction of Regulatory Subunits with the F1 Sector of ATP Synthase in Mitochondria

Mitochondrial ATP synthase has two stabilizing factors, 9K protein and 15K protein. The 9K protein was found to bind directly to F1-ATPase in the same way as ATPase inhibitor does. When 9K protein and the inhibitor were added simultaneously to F1-ATPase, they bound to the enzyme competitively, while when added successively with several minutes between their additions only the one added first bound tightly and it was scarcely replaced by the other added later. The F1-ATPase-9K protein complex retained about 60% of the ATP-hydrolyzing activity of the free enzyme and this activity was not inhibited by addition of the inhibitor. These characteristic binding features of the inhibitor and 9K protein were also observed with membrane-bound F1F0-ATPase unless the other stabilizing factor, 15K protein, was present. In the presence of 15K protein both the inhibitor and 9K protein could bind to F1F0-ATPase simultaneously but only one of the two ligands interacted directly with the F1 sector; exchange with the other ligand could not be achieved by the enzyme alone. These results indicated the existence of an assembly for regulating the activity of mitochondrial ATP synthase consisting of an inhibitor and stabilizing factors; the inhibitor and stabilizing 9K protein are counterparts in regulation of the enzyme activity, their bindings resulting in inactive and active F1, respectively.