Biosynthesis of glycogen in Neurospora crassa. Existence of a glucoproteic intermediate in the initiation process.

A soluble enzyme preparation (20,000 X g supernatant fraction), prepared from the mycelia of wild-type Neurospora crassa, was capable of transferring [14C]glucose from UDP-[14C]glucose into both trichloroacetic acid (TCA)-soluble and TCA-insoluble macromolecule products in the absence of added primer. These reactions did not require either high concentrations of salts or any other chemical reagents. Two labeled products were formed; one was a glycogen-like polysaccharide and the other was a glycoprotein with glucosyl chains bound to protein through an acid-labile bond. After mild treatment of the glucoprotein with acid, the product liberated from the protein behaved as a mixture of malto-oligosaccharides and alpha-1,4-glucan with branches. The carbohydrate moiety of the glucoprotein seemed to be released upon prolonged incubation with the enzyme preparation. The glucan thus liberated from the glucoprotein may serve as a primer for the glycogen synthase. The results obtained are therefore suggestive of the existence of a glucoproteic intermediate in the initiation of glycogen biosynthesis.     

Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes

The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.     

Biosynthesis of cytokinins by potato cell cultures

Potato cells grown in liquid culture incorporated mevalonic acid lactone-[2-¹⁴C] into free cytokinin (zeatin riboside and zeatin and the cytokinin of RNA (zeatin riboside). The cytokinin liberated by catabolism of RNA can account for no more than 40% of the free cytokinins.     

Biosynthesis of furano-terpenes by sweet potato cell culture

Callus was induced from sweet potato root tissue on an agarmedium containing Heller's minerals, vitamins, 2,4-D, yeastextract and sucrose. Furano-terpenes were scarcely detectedin the callus. However, when the callus was transferred to aliquid culture medium and incubated with reciprocal shaking,furano-terpenes were rapidly produced mainly in the culturemedium. Furano-terpene production by the cell culture was suppressedby addition of Ceratocystis fimbriata spores or HgCl₂ to theculture medium. Yeast extract and sucrose in the culture mediumwere important for furano-terpene production. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity increased in the cells,followed by the production of furano-terpenes. The TLC patternof furano-terpenes produced by the cell culture was essentiallythe same as that produced by sweet potato root tissue infectedby C. fimbriata or treated with HgCl₂, but the quantitativeproportion of the individual furano-terpenes in the former differedmarkedly from that in the latter. (Received January 11, 1979; )     

Deep-freezing of potato starch

Samples of oven-dried, air-dried, and moisturised potato starch (5, 13, and 24% w/w moisture content, respectively) were frozen in liquid nitrogen. Samples after thawing were studied by means of cross-polarised light beam microscope (CLBM), Fourier Transformation Infrared Spectroscope (FT-IR), powder X-ray diffractometer, and non-contact Atomic Force Microscope (nc-AFM). Rapid deep-freezing followed by thawing produced changes on the granule surface. They were accompanied by internal alteration manifested by FT-IR spectra and powder X-ray diffractograms. The results depended on the water content in the sample. Deep-freezing of moistened starch resulted in increased crystallinity of granules. It had minor effect on the granule aqueous solubility and characteristics of gelation.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.     

Kinetics of potato starch fermentation by Schwanniomyces castellii

The growth behaviour of Schwanniomyces castellii in slurry fermentation systems using untreated potato starch as substrate was studied in order to asses the eventual effect of the initial concentration of substrate (S_o) on cell growth rate. By applying the elementary balance method in combination with a Monod-type kinetic equation it was possible to formulate not only an unstructured model, but also the stoichiometry for such a yeast fermentation process. From a kinetic viewpoint, the Monod model was found to be redundant with respect to the pseudo-first order one, it being impossible to discriminate the contribution of v _M and K _S on the overall fermentation kinetics. Whereas the main yield coefficients appeared to be independent of S _O, the pseudo-first order rate constant was found to be inversely proportional to S _O. Therefore, cell growth appears to be controlled by the initial amount of amylolytic enzymes, that is to some extent proportional to the inoculum size, instead of the initial concentration of potato starch, at least within the experimental range of 3 to 30 g dm³.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.     

Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae.

Incubation of membranes prepared from Saccharomyces cerevisiae with [32P]phosphatidyl[3H]inositol resulted in the transfer of both labels to two products which were characterized as two species of inositolphosphoceramide, differing in the ceramide portion of the molecule. The products were characterized on the basis of stability in mild alkali, mobility on silica gel-impregnated paper, chromatography on silicic acid columns, and release of inositol phosphate upon base hydrolysis. The reaction did not require the addition of metals, nor was it inhibited by ethylenediaminetetraacetic acid. The detergents Triton X-100 and Tween 20 provided little, if any, stimulation. At relatively high concentrations of phosphatidylinositol (1 to 4 mM), the in vitro rate was about 20% of the in vivo rate. Although ceramide was a logical substrate, the reaction could not be greatly stimulated by the addition of ceramides containing mono- and dihydroxy fatty acids. In addition, incubation of yeast membranes with [32P]phosphatidylinositol gave rise to a product that was chromatographically indistinguishable from the major yeast phosphosphingolipid, mannose-(inositol-P)2 ceramide.     

Microwave-assisted silication of potato starch

Silication of potato starch was performed by microwave irradiation and convectional heating of starch with sodium metasilicate. The study has shown that microwaves offered more selective silication than convectional heating. Depending on the dose of metasilicate products of either monoesterification or crosslinking esterification were formed. Increase in the amount of the silicating agent favoured crosslinking of starch. In the case of microwave irradiation, the C–O–SiO₂Na moieties were formed, whereas the convectional heating generated the C–O–Si–O–Si–O–C crosslinks.     

Biosynthesis of chondroitin sulphate by a Golgi-apparatus-enriched preparation from cultures of mouse mastocytoma cells.

Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.     

Maturation and subcellular compartmentation of potato starch phosphorylase.

The subcellular localization and maturation of starch phosphorylase (EC 2.4.1.1) was studied in developing potato tubers. The enzyme is localized inside the stroma of amyloplasts in young tubers, whereas in mature tubers it is found within the cytoplasm in the immediate vicinity of the plastids. A phosphorylase cDNA clone was isolated and used in RNA gel blot experiments to demonstrate that phosphorylase mRNAs are of the same size and abundance in both young and mature tubers. In vitro translation of mRNAs followed by immunoprecipitation with a phosphorylase antiserum indicates that the enzyme is synthesized as a higher molecular weight precursor in both young and mature tubers. The presence of a transit peptide at the N terminus of the protein was confirmed by the sequencing of the phosphorylase cDNA clone. The transit peptide has several structural features common to transit peptides of chloroplast proteins but contains a surprisingly large number of histidine residues. The mature form of the enzyme is present in both young and mature tubers, suggesting that a similar processing of the transit peptide may take place in two different subcellular locations.     

Gelatinization mechanism of potato starch

The non-Newtonian behavior and dynamic viscoelasticity of potato starch (Jaga kids red ’90, 21.0% amylose content) solutions after storage at 25 and 4°C for 24 h were measured with a rheogoniometer. The flow curves, at 25°C, of potato starch showed plastic behavior >1.0% (w/v) after heating at 100°C for 30 min. A gelatinization of potato starch occurred above 1.0% at room temperature. A very large dynamic viscoelasticity was observed when potato starch solution (3.0%) was stored at 4°C for 24 h and stayed at a constant value with increasing temperature. A small dynamic modulus of potato starch was observed upon addition of urea (4.0 M) at low temperature (0°C) even after storage at 25 and 4°C for 24 h. A small dynamic modulus was also observed in 0.05 M NaOH solution. Possible models of gelatinization and retrogradation mechanism of potato starch were proposed.