Toshihide Takasawa

Chemical characterization of sialyl oligosaccharides isolated from tammar wallaby (Macropus eugenii) milk

Two components of the sialyl oligosaccharides were separated from milk of the tammar wallaby (Macropus eugenii) by gel filtration and ion exchange chromatography. Their molecular weights, estimated by gel filtration on HPLC using 5 mM triethylamine-acetate buffer (pH 5.0), were approx. 3,000. Their monosaccharide compositions, determined by GC analysis after methanolysis and by colorimetric assay, were (Glc)1(Gal)9(GlcNAc)2(Neu5Ac)1, and (Glc)1(Gal)8(GlcNAc)2(Neu5Ac)2. Their chemical structures were further elucidated by 1H-NMR and methylation analysis. The results suggest that their approximate structures are: [formula: see text]

Biosynthesis of (+)-cubenene and (+)-epicubenol by cell-free extracts of cultured cells of Heteroscyphus planus and cyclization of [^2H]farnesyl diphosphates

The absolute stereochemistry of cubenene and epicubenol from cultured cells of Heteroscyphus planus was determined as both (+)-isomers by 1H and 13C NMR spectroscopy, GLC using a chiral capillary column, and optical rotations. Incubation of two geometrical isomers of deuteriated farnesyl diphosphate (FPP) with a cell-free extract from cultured cells indicated that both compounds were specifically formed from (2E,6E)-FPP. Gas–liquid chromatography–mass spectrometry (GLC–MS) and 2H NMR analyses of (+)-cubenene and (+)-epicubenol generated from [1,1-2H2]- and [6-2H]-FPP confirmed the presence of 1,2- and 1,3-hydride shifts in their formation.

Lignan biosynthesis in liverworts Jamesoniella autumnalis and Lophocolea heterophylla

Abstract The formation of the lignans in liverworts, Jamesoniella autumnalis and Lophocolea heterophylla was investigated. Feeding experiment of |8- 2 H| caffeic acid ( 4 ) on axenic cultures of J. autumnalis has shown that jamesopyrone ( 2 ) and scapaniapyrone ( 3 ) were derived from the coupling of two intact caffeic molecules. Studies in a cell-free system of J. autumnalis and L. heterophylla have shown the formation of (±)- 1 from 4 .

Amino Acid Sequence of Acylphosphatase from Porcine Skeletal Muscle

The amino acid sequence of acylphosphatase from porcine skeletal muscle was determined. It consists of 98 amino acid residues with N-acetylserine at the amino (N)-terminus: Ac-Ser-Thr-Ala-Arg-Pro-Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly -Arg-Val-Gln-Gly-Val-Cys-Phe-Arg-Met-Tyr-Thr-Glu-Asp-Glu-Ala-Arg-Lys-Ile -Gly-Val-Val-Gly-Trp-Val-Lys-Asn-Thr-Ser-Lys-Gly-Thr-Val-Thr-Gly-Gln -Val-Gln-Gly-Pro-Glu-Glu-Lys-Val-Asn-Ser-Met-Lys-Ser-Trp-Leu-Ser-Lys -Ile-Gly-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Asn-Phe-Ser-Asn-Glu-Lys- Thr-Ile-Ser-Lys-Leu-Glu-Tyr-Ser-Asn-Phe-Ser-Ile-Arg-Tyr-OH. This sequence has three substitutions of amino acid residues, i.e., Thr/Ala, Ile/Val, and Ile/Val at positions 26, 68, and 96, respectively, from that of horse muscle acylphosphatase, formerly the only mammalian acylphosphatase with known sequence.

In vitro biosynthesis of cadinanes by cell-free extracts of cultured cells of Heteroscyphus planus

A cell-free extract from the calli of the liverwort Heteroscyphus planus catalyzes the divalent metal ion-dependent conversion of (2Z, 6E)-farnesyl diphosphate (FPP) into (-)-γ-cadinene and (+)-germacrene D, while it specifically converts (2E, 6E)-FPP into (+)-cubenene and (+)-epicubenol. The 1,3-hydride shift in the formation of (-)-γ-cadinene has been determined by conversion of (2Z, 6E)-[1,1-2H2]-FPP into (-)-γ-cadinene which was shown by GLC–MS and 2H NMR spectroscopy to be labelled at the C-11 position. These findings suggest that (-)-γ-cadinene is directly formed from 2Z, 6E-FPP by intramolecular electrophilic attack of the primary carbocation on the C-10 position of FPP. (+)-Cubenene synthase and (-)-γ-cadinene synthase has been purified by fractionation with ammonium sulfate, gel filtration on Sephacryl S-200 HR and anion exchange chromatography on DEAE-Sepharose CL-6B. Separation of (-)-γ-cadinene synthase from (+)-cubenene synthase is facilitated by a change in the elution behaviour of enzymes during anion exchange chromatography. All the evidence strongly suggests that the activities of (+)-cubenene synthase and (+)-epicubenol synthase are dual functions of the same enzyme.

Isolation of Three Creatine Kinases MM from Porcine Skeletal Muscle

The purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pI 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.