Yukio Hasegawa

Chemical structure of angiotensin in the bullfrog Rana catesbeiana

Crude bullfrog angiotensin was prepared by modified Bouchers procedures. The kidney extract was incubated in vitro with homologous plasma. Crude angiotension, 15 micrograms equivalent to [Asp1, Ile5]angiotensin II in rat vasopressor assay, was purified further by three steps by SEP-PAK C18 cartridge, SP-Sephadex column chromatography, and high-performance liquid chromatography on Finepak SIL C18 column. Amino acid analysis was done on 64 ng of the purified material after acid hydrolysis. The fluorescent peptide mapping technique was used to confirm the amino acid sequence. It is proposed that bullfrog angiotensin is [Asp1, Val5, Asn9]angiotensin I.

Determination of inorganic phosphate by flow injection method with immobilized enzymes and chemiluminescence detection

A flow injection method with chemiluminescence detection has been developed for the enzymatic determination of inorganic phosphate. Purine nucleoside phosphorylase, xanthine oxidase, and urate oxidase were immobilized on controlled-pore glass beads. Hydrogen peroxide released by the enzymatic reactions of phosphate and inosine in carrier buffer was detected by the luminol-microperoxidase system in a flow cell. The calibration graph was linear over the range of 5 to 250 pmol, and reproducibility was 1.75% at 10 pmol. The detection limit was 500 fmol of phosphate in 50 microliters of sample injected. The phosphate content in deoxyribonucleic acid was measured by this method.

Chemical structures of angiotensins formed by incubating plasma with the kidney and the corpuscles of Stannius in the chum salmon, Oncorhynchus keta

The chemical structures of salmon angiotensins produced by incubating tissue extract of the kidney or the corpuscles of Stannius (CS) with homologous plasma are proposed. Two angiotensins, [Asp1, Val5, Asn9] and [Asn1, Val5, Asn9] angiotensin I, were proposed from both kidney and CS incubations by amino acid analysis and the fluorescent peptide-mapping techniques. CS angiotensins were not organ specific, because these two angiotensins were produced by both kidney and CS incubations in a ratio of 1:2 under the same conditions. Whether [Asp1, Val5, Asn9] angiotensin I is a naturally occurring form remains to be clarified; however, [Asn1, Val5, Asn9] angiotensin I may be the major form of angiotensin formed from plasma by salmon kidney and CS.

Chemical structure of angiotensin in the turtle, Pseudemys scripta

The chemical structure of angiotensin generated by incubating kidney extract with homologous plasma from the turtle, Pseudemys scripta, has been analyzed. The turtle angiotensin was proposed to be [Asp1, Val5, His9] ANG I by its amino acid composition and by its fluorescent peptide mapping. It was the same structure as angiotensin I in the ox and the sheep. The N-terminal amino acid of the turtle angiotensin was not blocked, unlike that of another reptilian angiotensin found in the snake, Elaphe climacophora.

Vasopressor and depressor effects of native angiotensins and inhibition of these effects in the Japanese quail

The amino acid sequence of angiotensin I (ANG I) from the Japanese quail, Coturnix coturnix japonica, obtained from incubation of homologous plasma and kidney extract was determined as H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ser-Leu-OH. A bolus, intravenous injection of native ANG I or of ANG II induced an immediate vasodepressor response and a subsequent vasopressor response in quail which has been lightly anesthetized with urethane (0.75 g/kg). The values for ED50 for the vasopressor and depressor effects of ANG II were 85 and 113 pmol/100 g body weight, respectively. The extent of the hypotension was dependent on the arterial pressure prior to injection. The effects of ANG I and II on heart rate were variable. Human [Asp1, Ile5, His9] ANG I and II were almost as potent as their quail counterparts with respect to the cardiovascular effects, but eel [Asn1, Val5, Gly9]ANG I and II were less than half as potent. Human ANG III had little effect on arterial pressure in the quail. A bolus injection (100 micrograms/100 g) or infusion (1 micrograms/100 g/min) of [Sar1, Ile8] ANG II almost abolished the cardiovascular effects of ANG I and II, but [Sar1, Ala8] ANG II and [Sar1, Thr8] ANG II, which are effective inhibitors in mammals, had no inhibitory effects. The vasopressor and depressor effects of ANG I were abolished, while those of ANG II were slightly enhanced, after injection (100 micrograms/100 g) or infusion (1 micrograms/100 g/min) of SQ14225, whereas des-Pro2 bradykinin and bradykinin potentiator B, which are effective inhibitors of ANG I converting enzyme in mammals, failed to inhibit the effect of ANG I. These results indicate that vascular ANG II receptors and ANG I converting enzyme in the quail may be different from those in mammals.

Chemical structure of angiotensin formed by incubating plasma with the corpuscles of Stannius in the Japanese goosefish, Lophius litulon.

The chemical structure of angiotensin formed by incubating the extract of the corpuscles of Stannius (CS) with homologous plasma in the Japanese goosefish , Lophius litulon , was analyzed. Goosefish CS angiotensin was proposed as [Asn1, Val5, His9 ] angiotensin I based on amino acid analysis and fluorescent peptide mapping techniques. It had the same structure as angiotensin produced by incubating the kidney extract with homologous plasma, indicating that CS angiotensin was not organ specific.

Determination of platelet-activating factor by a chemiluminescence method and its application to stimulated guinea pig neutrophils

A simple, rapid and sensitive chemiluminescence method has been developed to measure platelet-activating factor (PAF). Hydrogen peroxide generated from PAF, upon phospholipase D cleavage, by choline oxidase is determined as chemiluminescence by a luminol-microperoxidase system. The detection limit of PAF by this method is 5 pmol/tube. The method is reproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n=5). Lipids were extracted from guinea pig neutrophils after stimulation with cytochalasin B andN-formyl-methionyl-leucylphenylalanine, and PAF was isolated by high-performance liquid chromatography and determined by chemiluminescence measurements. The amount of PAF detected was 96.1±39.7 (mean ± SD, n=7) pmol/108 cells. This highly sensitive method could be useful for the determination of PAF generated under pathophysiological conditions.