Spleen phospholipases A2.
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Amino Acid Sequence↗
Biomedical subjects
H Tojo
Publications and source records attributed to H Tojo.
Immunochemical relationship of D-amino acid oxidases in various tissues and animals.
1. By means of an enzyme immunoassay, the contents of D-amino acid oxidase (DAO) were determined in kidney, liver, cerebellum and lung of hog, but the oxidase was not detectable in heart or cerebrum. 2. The oxidases in kidney, liver and cerebellum of hog were indistinguishable as regards immunoreactivity toward anti-hog kidney DAO antibody, specific activity and molecular weight. 3. The oxidases in rat and dog kidneys immunochemically cross-reacted with anti-hog DAO antibody. 4. The overall structure of the hog oxidase was more similar to that of the dog enzyme than that of the rat, while the structure around the catalytic site of the hog oxidase was more similar to that of the rat oxidase than that of the dog enzyme. 5. On immunoblot analysis, two forms of the oxidase were detected in extracts of hog, rat and dog kidneys.
Animals↗
The existence of two different forms of apo-electron-transferring flavoprotein.
The association process of FAD and apo-electron-transferring flavoprotein (apoETF) from hog kidney was investigated. The reaction schemes which involve the association-dissociation of the protein species could be excluded by the light scattering data, which indicated that the molecular weights of apoETF and holoETF are identical. The binding reaction between FAD and a large excess of apoETF was monophasic and obeyed pseudo-first order kinetics. On the other hand, the reaction between apoETF and a large excess of FAD was biphasic: the fast phase obeyed a pseudo-first order reaction, and the rate of the slow phase was almost independent of FAD concentration. These results suggest the existence of two different forms of apoETF, as represented in the following reaction scheme: [formula: see text] where "F" is FAD, "H" is holoETF, and "A" and "A" are the different forms of apoETF. The kinetic parameters were determined as k-1 = 3.9 x 10(4) M-1.s-1, k-1 approximately 10(-5) s-1, k+2 = 1.0 x 10(-3) s-1, and k-2 = 3.1 x 10(-3) s-1, in 50 mM potassium phosphate buffer, pH 7.6, containing 0.3 mM EDTA, and 5% v/v glycerol, at 7 degrees C. The elution patterns of apoETF on molecular sieve chromatography were very different from that of holoETF although the true molecular weights were identical. This result suggests that the structure of apoETF differs greatly from that of holoETF.
Animals↗
[A rapid method for screening transgenic mice by PCR with small amounts of blood].
We report a rapid method for screening transgenic mice by polymerase chain reaction (PCR) with small amounts of blood. Ten to 50 microliters of the uncoagulated blood were collected from the transgenic mice carrying the human A gamma/beta-globin gene (8.9 kb, 13 and 53 copies) by puncturing the leg vein or cutting the tail end. Blood was mixed well with 200 microliter of the white blood cell separating-solution and was stood for 40 minutes. DNA was extracted from the blood cells in the supernatant and was used for PCR. When 10-20 microliters of blood were applied to the cell separating-solution and the blood cells that washed out from 10-20 microliters of the supernatant were used for PCR, the specific DNA band that amplified from the transgene was always detected by the electrophoresis with an agarose minigel.
Animals↗
Effects of dietary zinc and cadmium on tissue selenium concentration and glutathione peroxidase activity in rats fed DL-selenomethionine or sodium selenite.
The effects of dietary zinc (Zn) and cadmium (Cd) on tissue selenium (Se) concentration and glutathione peroxidase (GSH-Px) activity were studied in weanling male Wistar rats. One group of rats was fed a purified diet based on casein and sucrose, and the other rats used in a 2 x 2 x 2 factorial arrangement of treatment were fed this diet supplemented with 0.1 mg Se/kg, either as DL-selenomethionine or sodium selenite and plus 100 mg Zn/kg as zinc sulfate or 5 mg Cd/kg as cadmium chloride or both for 4 weeks. Se concentrations in plasma, erythrocytes, muscle, heart, and liver were significantly elevated by Zn. Cd significantly decreased Se concentration in muscle. Addition of Zn to the diets markedly increased (p less than 0.001) hepatic GSH-Px activity. However, Cd in the diets produced a significant increase (p less than 0.001) in erythrocyte GSH-Px activity. These results indicate that Zn level of marginal deficiency (8.6 mg/kg diet) can decrease Se availability and a small excess of Zn increases Se availability for hepatic GSH-Px activity.
Animals↗
Fatty acid composition of breast milk from Nigerian and Japanese women.
The fatty acid composition of samples of breast milk obtained from well-nourished Nigerian and Japanese women was determined by gas chromatography. The cultural differences in dietary intake was reflected in the fatty acid composition of breast milk samples. The milk of Nigerian women contained a significantly higher percentage of saturated fatty acids (48.75%) than that of Japanese women (46.65%). Nigerian milks were also richer in arachidonic (20:4 n-6), eicosatrienoic (20:3 n-6), and docosatetraenoic (22:4 n-6) acids. Conversely, the milk of Japanese woman contained significantly higher percentages of monoun-saturates as palmitoleic, heptadecenoic, oleic, and polyunsaturates of n-3 series as alpha-linolenic, eicosapentaenoic, and docosahexaenoic acid.
Adult↗
The necessity of dietary vitamin B6 to selenium biopotency for tissue selenium and glutathione peroxidase in rats.
Necessity of dietary vitamin B6 to the biopotency of selenium (Se) for the levels of Se and glutathione peroxidase (GSH-Px) in tissues was investigated. Male Wistar 12-week-old rats were fed a vitamin B6-Se-deficient basal diet for 3 weeks, and then the rats were divided into 6 groups. One group was fed the basal diet, the others were fed the diet supplemented with 250 micrograms vitamin B6/100 g as pyridoxine.HCl, or 0.25 mg Se/kg as Na2SeO3 (SeL) or DL-selenomethionine (Se-Met), or both (SeL+B6 or Se-Met+B6) for 10 week. The levels of Se and GSH-Px in erythrocytes and muscle were significantly higher in vitamin B6-supplemented groups than in vitamin B6-deficient groups. There was little effect of this vitamin deficiency on Se level in liver of rats fed SeL; however, a higher Se level in liver was observed in vitamin B6-deficient rats fed Se-Met than in the corresponding B6-supplemented rats. A significant decrease of GSH-Px activity in liver was found in vitamin B6-deficient animals fed Se-Met compared with vitamin B6-supplemented animals, whereas no significant decrease was observed in those fed SeL. These results suggest that this vitamin is involved in the transport and deliverance of Se in plasma to the other tissues and the incorporation of Se from Se-Met to GSH-Px in liver.
Animals↗
[Structure and function of phospholipases A2 from the digestive system and spleen].
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Acute Disease↗
Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells.
Using a specific polyclonal antibody raised against rat pancreatic phospholipase A2 (PLA2), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA2 reactive with the anti-pancreatic PLA2 antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA2 and PAS was observed in the same cell, it is suggested that PLA2 could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA2 in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA2 secreted from the pancreas. Other possible roles of the PLA2 in the stomach are discussed.
Animals↗
[Evaluation of separating X- and Y-sperms by percoll density gradient centrifugation using sperms of transgenic mice carrying a transgene on Y-chromosome].
Using sperms of the transgenic mice carrying a human A gamma/beta-globin gene on Y-chromosome, we attempted to separate X- and Y-bearing sperms by the Percoll density gradient centrifugation. The ratio of X- and Y-sperms was determined by DNA dot blot hybridization procedure with sperm DNA. Sperm suspension collected from cauda epididymidis was loaded on the gradient composed of 7 Percoll concentrations (35-84%) and was centrifuged at 300 x g for 10, 15 or 20 minutes, respectively, at room temperature. After centrifugation, sperms were collected from each gradient fraction and washed with 0.85% saline solution. DNA was extracted from sperms, dotted and fixed on nitrocellulose filter, and was hybridized with the 32P-labeled DNA probe derived from the beta-globin gene. Each DNA spot was cut out, immersed in the liquid scintillator and was counted for radioactivity. There was no difference among the radioactivities in the DNA spots, indicating that the ratio of X- and Y-sperms was the same in all the gradient fractions of three different centrifugal conditions. The results suggests to be difficult to separate X- and Y-sperms by Percoll density gradient centrifugation, at least, using sperms from cauda epididymidis of mouse.
Animals↗
Effect of fish oil and safflower oil in common Japanese diet on human plasma fatty acid composition.
The influence of fish oil and safflower oil contained in the common Japanese diet as the main dietary polyunsaturated fatty acid source on plasma fatty acids in ten female student volunteers (21-22 years old) was investigated. The subjects were divided into two groups and fed the experimental diets for five days. The total daily fat intake in the fish diet and safflower oil diet was 54.4 g and 56.2 g, respectively, and the fat derived from fish and safflower oil was 16 g and 23 g, respectively. The proportion of linoleic acid was reduced in the plasma of subjects fed the fish diet and increased in the plasma of subjects fed the safflower oil diet. The plasma levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were significantly elevated in the fish diet group. The ratio of EPA/arachidonic acid (AA) was higher, and those of n-6/n-3 and n-9/n-3 were lower in the plasma of subjects fed the fish diet when compared to the results obtained from plasma of subjects fed the safflower oil diet. From these results, it seems likely that fish oil in the common Japanese diet is a favorable source of plasma EPA and DHA even in such short term supplementation and with such a small amount of daily consumption.
Adult↗
[Myocardial injury and phospholipases].
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Acyl Coenzyme A↗
The primary structure of a membrane-associated phospholipase A2 from human spleen.
The complete primary structure of membrane-associated phospholipase A2 purified from a human splenic membrane fraction was determined by sequence analysis of the peptides generated by lysyl endopeptidase and Staphylococcus aureus V8 protease cleavage. The enzyme consists of 124 amino acid residues corresponding to a molecular weight of 13,904. The primary structure reveals the characteristics of Group II phospholipases A2 and a large ratio of basic amino acid residues to acidic ones, that ratio being 3.4 : 1.
Amino Acid Sequence↗
cDNA cloning and sequence determination of rat membrane-associated phospholipase A2.
Based on the partial amino acid sequences of membrane-associated phospholipase A2 (PLA2M), belonging to group II, purified from rat spleen, the cDNA encoding PLA2M was cloned by a new cloning strategy utilizing enzymatic cDNA amplification. At the N-terminus of the coded 146 residues, which were deduced from the cDNA sequence, the putative signal peptide was found despite the tight adherence of this enzyme to the membrane. The sequence of rat PLA2M exhibits 75% homology with that of human group II PLA2 in the protein-coding region. The result of RNA blot analysis showed that rat ileal mucosa contains the largest amount of the PLA2 transcript among the tissues examined.
Amino Acid Sequence↗
Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung.
The content of mRNA for a pancreatic-type phospholipase A2 present in rat gastric mucosa was much greater than that in pancreas. In lung the mRNA for this pancreatic-type phospholipase A2 was also detected, but less than in pancreas. Nucleotide sequence analysis showed that these pancreatic-type phospholipase A2 cDNAs derived from rat gastric mucosa and lung were completely identical to that from rat pancreas (Ohara et al. (1986) J. Biochem. 99, 733-739). This demonstrates that the pancreatic-type phospholipase A2 present in gastric mucosa and lung does not originate from pancreas.
Animals↗
A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2.
Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.
Amino Acid Sequence↗
Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1.
A membrane-associated phospholipase A2 was purified from rat spleen. The phospholipase A2 was solubilized from the 108,000 x g pellet fraction with 0.3% lithium dodecyl sulfate and then purified to homogeneity by successive DEAE-Cellulofine AM, octyl-Sepharose, Cellulofine GCL 300-m, S-Sepharose, and Bio-Gel P-30 chromatographies in the presence of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate. The apparent Mr of the enzyme, estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, was about 13,600. The purified enzyme had a pH optimum in the range of pH 8.0-9.5 and required the presence of Ca2+ (4 mM) for its maximal activity. The enzyme preferentially hydrolyzed the 2-acyl ester bonds of phosphatidylglycerol in the presence and absence of sodium cholate or sodium deoxycholate. Unlike the phospholipase A2 of rat spleen supernatant, no immunocross-reactivity was observed between the purified enzyme and anti-rat pancreatic phospholipase A2 antibody. The N-terminal amino acid sequence of the enzyme was determined and found to be homologous to that of viperid and crotalid venom phospholipases A2. The results in this and the preceding report (Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731) demonstrate that rat spleen contains two genetically distinct phospholipase A2 isoenzymes.
Amino Acid Sequence↗
A pancreatic-type phospholipase A2 in rat gastric mucosa.
A phospholipase A2, which is immuno-crossreactive with the anti-rat pancreatic phospholipase A2 antibody, is present in rat gastric mucosa. The content of the enzyme in the gastric mucosa was comparable to that in the pancreas, but the specific activity in the gastric mucosa homogenate (60.7 +/- 19.5 nmol/min/mg) was higher than that in the pancreas homogenate (3.16 +/- 0.77 nmol/min/mg). A greater proportion of the enzyme was found in the particulate fraction. The gastric enzyme and its proenzyme were purified from the supernatant. The amino acid sequence of the N-terminal 15 residues of the gastric enzyme was determined and found to be identical with that of rat pancreatic phospholipase A2. Like the pancreatic proenzyme, the gastric proenzyme was activated on trypsin treatment.
Animals↗