Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Characterization of O-trimethylsilyl derivatives of d-glucose,d-galactose,and d-mannose by gas-liquid chromatography-chemical-ionization mass spectrometry with ammonia as reagent gas

The per(trimethylsilyl) ethers of d-glucose, d-galactose, and d-mannose were analyzed by g.l.c.-c.i.m.s. with ammonia as the reagent gas. C.i.m.s. gave simple fragmentation and fragment ions of high intensity in the high-mass range where the QM⁺ ion is also detected. The β-d anomers gave ions at m/e 558 showing intensities 3–12 times those of the α-d anomers. The epimers could be distinguished by differences in the intensities of the ions and by the observation that d-glucose gave a base peak at m/e 198, d-galactose at m/e 468, and d-mannose at m/e 204. The pyranose and furanose structures could be distinguished by comparing the ion intensities at m/e 198, m/e 271, m/e 361, m/e 396, and m/e 451. A similar analysis was also performed with 2-methylpropane as the reagent gas.     

Presence of a xyloglucan in the cell wall of Phaseolus aureus hypocotyls

The non-cellulosic ß-glucan₁ in the cell wall of Phaseolusaureus hypocotyb was studied. Evidence that xyloglucan is presentin a hemicellulose fraction was obtained by its isolation fromcell wall preparations. This polysaccharide was homogeneouson zone electrophoresis and ultracentrifugation. On acid hydrolysis,it gave glucose, xylose, galactose, and fucose in the approximatemolar ratio of 10 : 7 : 2.5 : 1. Its solution gave a reddishviolet color with iodine-staining solution. The results of partialacid hydrolysis and cellulase treatment suggest a structurein which xylose, galactose, and fucose attached as side chainsto a sequenceof ß-l,4-linked glucose. The xyloglucanisolated accounted for 13.9% of the total non-cellulosic fractions. (Received May 10, 1976; )     

Two-nucleotide codon change in a hemoglobin polymorphism of the Celebes black ape (Macaca nigra)

A hemoglobin polymorphism involving variant -chains was demonstrated in the Celebes black ape, Macaca nigra. Fingerprinting and amino acid analysis of the tryptic peptides from the two chain types have shown that they differ by a single amino acid substitution, between lysine and aspartic acid, which requires a two-nucleotide change in the corresponding codon. Another substitution in the same codon is found as a species difference between the black ape -chain and that of other macaques.     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch I. Its isolation and some enzymic properties

A method is described for isolating phosphomannose isomerasefrom young konjak corms. The enzyme is believed to catalyzethe mannose forming reaction in growing corm tissues. The purifiedenzyme preparation was free from phosphoglucose isomerase activity,and was stable at pH 6–9. Maximum enzyme activity wasobserved at pH 6.5–7.0. The molecular weight of the enzymewas estimated as 45,000 using Sephadex gel filtration. The followingkinetic parameters were obtained: Km (mannose-6-P), 0.73 mM,K_eq (fructose-6-P/mannose-6-P), 1.06 at pH 6.5, and activationenergy, 11,600 cal/mole. The enzyme was inhibited by the metalbinding agents EDTA, o-phenanthroline, and a,a'-bipyridyl. Theinhibitory effect of these agents was markedly influenced bythe pH level of the incubation mixture, being more pronouncedat pH 6 than at pH 8. ¹ This paper constitutes part 3 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Influence of Dietary Condition on Muscle Protein Composition

Contents of arginine, methionine, histidine, phenylalanine, tryptophan and tyrosine were measured in the “Extract” and the “Fibrous” proteins obtained by urea fractionation from the skeletal muscle of rats fed a normal, a tryptophan-free and a protein-free diets for 30 days respectively. The ratios of the “Extract” to the “Fibrous” proteins were remarkably different in the muscles of the different dietary groups. Significant differences, were observed in the amino acid compositions of the protein fractions between the normal and both deficient groups showing the presence of the different dietary effects on the constituent proteins of the muscle.

A quantitative fractionation method of muscle protein was presented. Muscles from the adult rats fed a normal, a tryptophan-free and a protein-free diets separately for 4 weeks were fractionated by this method. The effects on the composition of the muscle protein fractions were different between the tryptophan-free and the protein-free diets. The decreases of non-protein nitrogen and sarcoplasmic protein by both deficient diets were greater than that of total muscle nitrogen, whereas those of actin, myosin and stroma were smaller. This shows the presence of the different dietary effects on the constituent proteins of muscle.