Biomedical subjects
Publications and source records attributed to R Sato.
S-Methylmethionine (vitamin U) markedly increased the fecal excretion of both 14C-bile acids and 14C-neutral sterols in 14C-cholesterol injected mice, and did not inhibit both intestinal absorption of 14C-cholesterol and biosynthesis of 14C-cholesterol from 14C-acetate in mice.
Resonance Raman spectra have been measured for cytochromes P-450 purified from liver microsomes of phenobarbital-treated rabbits (PB P-450 and PB P-448) and of 3-methylcholanthrene-treated rabbits (MC P-448). In the reduced state, all three cytochromes P-450 exhibit Raman spectra of ferrous high-spin type but show the so-called "oxidation state marker" (band IV) at unusually low frequencies, indicating extensive delocalization of electrons from the iron dpi orbital to the porphyrin II (eg) orbital and, consequently, the strong pi basicity of the fifth ligand of the heme iron. The reduced CO complexes of the cytochromes P-450 also exhibit band IV at markedly lower frequencies than CO complexes of hemoglobin and myoglobin. These anomalies observed for the reduced form and CO complex disappear upon conversion of the cytochromes to the catalytically inactive form called cytochrome P-420. Oxidized PB P-450 shows a Raman spectrum which is characteristic of typical ferric low-spin heme compounds, whereas those of PB P-448 and MC P-448 are of the ferric high-spin type. PB P-450 is also clearly distinguishable from the two P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits two sets of Raman lines and, therefore, seems to be a mixture of both high- and low-spin species.
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Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.
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A method is described for the isolation of morphologically well-preserved Golgi apparatus from rat liver. The method is essentially the same as that of Morré et al. (Morré, D.J., Hamilton, R.L., Mollenhauser, H.H., Mahley, R.W., Cunningham, W.P., Cheetham, R.D., & Lequire, V.S. (1970) J. Cell Biol. 44, 484-491) except that mild cell disruption is achieved by means of a stainless-steel sieve. The average recoveries of protein and galactosyltransferase in the isolated fraction are about 6 mg from 10 g of perfused liver and about 35% from the homogenate, respectively. The preparation is virtually free from succinate-cytochrome c reductase, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase. The Golgi fraction as well as its vesicular fragments is homogeneous upon isopycnic centrifugation in both sucrose and dextran density gradients. Their buoyant densities in sucrose are significantly higher than those in dextran, indicating that both forms of the organelle are closed systems which are impermeable to macromolecules. The galactosyltransferase activity of a freshly prepared Golgi fraction, measured with ovalbumin as galactosyl acceptor, is activated 26-fold by the addition of Triton X-100, whereas those of homogenized, sonicated, and aged preparations are only activated 2- to 4-fold.
Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.
Vesicular fragments of Golgi apparatus, smooth- and rough-surfaced microsomes from rat liver are differently partitioned in aqueous polymer two-phase systems consisting of dextran, polyethylene glycol, and sodium phosphate buffer. At a given polymer concentration, the amount of material partitioned in the top phase increases in the following order: rough microsomes less than smooth microsomes less than Golgi fragments. Counter-current distribution of Golgi fragments in the system consisting of 6.8% (w/w) dextran T500 and 6.8% polyethylene glycol 4,000 results in the separation of the fragments into three fractions; i.e. Fractions I, II, and III. NADH- and NADPH-cytochrome c reductase activities are detected almost exclusively in Fraction I, whereas the activities of galactosyltransferase, acid phosphatase, 5'-nucleotidase, and thiamine pyrophosphatase are maximal in Fraction III and minimal in Fraction I. The distribution of these enzymes suggests that Fraction I is similar to, though not identical with, microsomes, Fraction III resembles plasma membrane and lysosomes, and Fraction II is between the two. It is concluded that NADH- and NADPH-cytochrome c reductases are localized in a restricted region of the Golgi structure and that intra-Golgi differentiation seems to proceed in a discontinuous manner.
The effects of various treatments in vivo on the intracellular contents of the Golgi apparatus and microsomes (endoplasmic reticulum) in rat liver were studied. Partial hepatectomy increased the content of Golgi apparatus. Laparotmy also increased the content of Golgi apparatus, but to a lesser extent than partial hepatectomy. In contrast, the content of microsomes remained unchanged by these treatments. On the other hand, the plasma seromucoid content was markedly increased by laparotomy, but unchanged by partial hepatectomy. Papain administration also caused an increase in the content of Golgi apparatus. The contents of both organelles were increased by the injection of phenobarbital. These results indicate that the control mechanisms of proliferation of Golgi apparatus are different from those of endoplasmic reticulum. These findings are discussed in relation to the functions of the Golgi apparatus, and it is suggested that the major function of the organelle at a given time is determined by the major metabolic demands at that time.
Rabbit cytochrome b5 was incorporated into single-walled phosphatidylcholine liposomes, and the cytochrome b5-liposome complex thus formed was digested with trypsin. Protein chemical characterization indicated that the main products formed were 1) a hydrophilic (heme-containing) fragment of the cytochrome corresponding to the sequence consisting of the masked NH2-terminus through residue 88, 2) a hydrophobic peptide which spans residue 91 to the COOH-terminus (residue 133), and 3) a dipeptide, seryl-lysine, derived from residues 89 and 90. The hydrophobic peptide was obtained in the form of its complex with liposomes. It was concluded that trypsin cleaved rather specifically the peptide bonds between residues 88 and 89 (Arg-Ser) and between residues 90 and 91 (Lys-Leu). Tryptic digestion of free, unbound cytochrome b5 also resulted in the cleavage of the same peptide bonds. These results are not consistent with the proposal of Visser et al. (Visser, L., Robinson, N.C., & Tanford, C. (1975) Biochemistry 14, 1194-1199) that the hydrophilic and hydrophobic domains of cytochrome b5 are connected to each other by a link peptide consisting of some 15 amino acid residues and that this link peptide can be cut out by the action of trypsin. The circular dichroism spectrum of intact cytochrome b5 or its complex with liposomes in the far-ultraviolet region was closely similar to the sum of the spectra of the hydrophilic fragment and the hydrophobic peptide (or its complex with liposomes). This indicates that the tryptic cleavage of the cytochrome molecule does not induce any significant changes in the conformations of the hydrophilic and hydrophobic moieties of the molecule and thus provides further evidence that the three-dimensional structures of the two domains are independent of each other.
Elucidation of mechanisms of angiotensin tachyphylaxis will be necessary for explaning the continuous vasocontricting action of angiotensin in the vascular smooth muscle, which might be closely correlated to the high blood pressure in renin-dependent or vasoconstricting hypertension. From the results obtained sodium loading rats showed not only augmented pressor response to angiotensin but also inhibiting the appearance of its tachphylaxis under the infusion of Ca-antagonist dose enough to suppress the response to angiotensin in the rats of water drinking. Speculated mechanisms about this phenomenon has been discussed.
A case of aortic regurgitation observed in a thalidomide deformed child is presented. Possibility of thalidomide as an etiologic factor was discussed.
The effects of acute and chronic administration of D-Galactosamine (GalN), Ethanol and Phenobarbital were investigated on the activities of lysosomal enzymes, i.e.; acid phosphatase, beta-glucuronidase and n-acetyl-beta-glucosaminidase, and others such as gamma-GTP and adenosine triphosphatase. The histochemical distribution of gamma-GTP in the liver was also studied on biopsy specimens from patients with chronic hepatitis, and gamma-GTP levels in the serum of patients receiving drugs inductable of hepatic microsomal enzymes. 1) After a single intraperitoneal injection of GalN, the lysosomal enzyme activities were lowered in the necrotic areas, but raised in the perinecrotic areas, the proliferative Kupffer cells and intra- and/or extra-cellular eosine bodies. 2) gamma-GTP activities in rat liver after chronic administration of GalN were markedly increased in bile canalicular membrane of periportal parenchymal cells, the epithelium of bile duct and ductules, and som inflammatory cells of portal fields. Levels of serum gamma-GTP were also elevated. On histochemical studies with biopsy specimens from patients with chronic active hepatitis showing elevated gamma-GTP activity, the activity was revealed a similar localization to GalN-treated rats. These data suggested that the increased activities might be reflected on the active stage in chronic hepatitis. 3) Chronic ethanol treatment in rats induced clearly-stained lysosomes varied in size, especially large-sized. The activities of hepatic gamma-GTP were slightly increased in the bile canalicular membrane of periportal parenchymal cells and the epithelium of proliferative bile ductules. 4) It has been shown by histochemical and biochemical techniques that hepatic gamma-GTP activity was increased after phenobarbital administration in rats. A significant rise in serum gamma-GTP was observed in patients on long-term treatment with anti-epileptic drugs. These data indicated that the increased activities of serum gamma-GTP might be accompanied with induction of hepatic microsomal drug-metabolizing enzymes.
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