A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.

Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.     

The use of fluorescamine as a probe for labeling the outer surface of the plasma membrane

A rapid method was developed to label the outer surface of chick embryo fibroblasts with fluorescamine without disruption of the cell monolayer. Polyacrylamide gel electrophoresis resolved two distinct areas of fluorescence: a group of high molecular weight polypeptides and several rapidly migrating species. The latter were demonstrated by tlc to be phospholipids. Fluorescamine did not label internal components of the cell as evidenced by two intracellular proteins which were found to be non-fluorescent. Intact normal cells were labeled 3-fold more than transformed cells, indicating a possible loss of exposed sites at the surface, while disrupted cells, subsequently labeled, yielded similar amounts of fluorescence.     

Preparation of cellulose–starch composite gel and fibrous material from a mixture of the polysaccharides in ionic liquid

This paper reports the preparation of a cellulose–starch composite gel from an ionic liquid solution. The gel was obtained by keeping the homogeneous mixture of cellulose (10% w/w) and starch (5% w/w) in 1-butyl-3-methylimidazolium chloride (BMIMCl) for several days at room temperature and characterized by elemental analysis, X-ray diffraction, and thermal gravimetric analysis. Furthermore, the production of the fibrous material composed of cellulose and starch by reconstitution from the homogeneous mixture (10% w/w each) in BMIMCl is demonstrated.     

Synthesis of sorbent from cross-linked starch for affinity purification of glucose(mannose)-specific lectins]

Cross-linked starch gel for the affinity chromatography of D-glucose (D-mannose)-specific lectins is suggested. In order to optimize hydrodynamic properties of gel 30% starch has been hydrolysed by HCI at 70 degrees C during 60 min and then cross-linked by epichlorohydrin under alkaline conditions. Every 100 g of starch require 18 ml of epichlorohydrin and 36 ml of 8 N KOH. The gel obtained has been successfully used for the purification of lectins from Pisum sativum L., Lens culinaris L., Vicia sativa L., and Vicia faba L. seeds. These lectins, purified on starch gel do not differ from sephadex-purified samples.     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

Effect of waxy rice flour and cassava starch on freeze-thaw stability of rice starch gels

Repeatedly frozen and thawed rice starch gel affects quality. This study investigated how incorporating waxy rice flour (WF) and cassava starch (CS) in rice starch gel affects factors used to measure quality. When rice starch gels containing 0-2% WF and CS were subjected to 5 freeze-thaw cycles, both WF and CS reduced the syneresis in first few cycles. However CS was more effective in reducing syneresis than WF. The different composite arrangement of rice starch with WF or CS caused different mechanisms associated with the rice starch gel retardation of retrogradation, reduced the spongy structure and lowered syneresis. Both swollen granules of rice starch and CS caused an increase in the hardness of the unfrozen and freeze-thawed starch gel while highly swollen WF granules caused softer gels. These results suggested that WF and CS were effective in preserving quality in frozen rice starch based products.     

Disc electrophoretic studies of hemoglobin dissociation by p-hydroxymercury benzoate

The reaction of excess p-mercurybenzoate with human hemoglobin produces five electrophoretic species on polyacrylamide gel. Only two of these bands have been previously observed when starch gel was employed. The chemical and electrophoretic studies presented in this paper illustrate that the appearance of an “extra” band in the β zone is due to the ability of PAGE to separate the βb₂^PMB ? β^PMB equilibrium to its discrete components. The remaining bands are accounted for by the superior resolution of PAGE over starch gel electrophoresis which allowed the detection of two (αβ)^PMB dimer species.     

Asymmetric Membrane Distribution of Phosphatidylserine in Different Liposomal Preparations

Abstract

We established a quantitative assay (fluorescamine-assay) to determine the distribution of phosphatidylserine (PS) in the outer and inner monolayer of mixed phosphatidylcholine/phosphatidylserine liposomes. Fluorescamine (a non-fluorescent compound) reacts with primary amino groups resulting in a fluorescent product. Fluorescamine is not able to penetrate through the liposomal membrane so only the amount of PS located in the outer monolayer is accessible for the reaction. This assay was used to study the influence of several factors in liposome preparation on liposome asymmetry. An increase in the amount of PS in the liposomal preparation leads to a decrease in the outer to inner PS ratio. This was also the case for unilamellar vesicles of decreasing size. The asymmetry of PS is only slightly influenced by the addition of phos-phatidylinositol or phosphatidylgycerol, whereas small amounts of phosphati-dylethanolamine-rhodamine-B induce a dramatic shift of PS to the inner monolayer. NBD-labeled PS shows a asymmetric distribution different from that of unlabeled PS.     

A study on the in vivo digestibility of retrograded starch

The digestibility of different forms of starch was examined in an ileostomy model. Six otherwise healthy ileostomists were fed a controlled polysaccharide-free diet for four days, on three of which a test starch was added at breakfast. 50 g starch was fed as either whole or homogenized chick peas or as a retrograded starch gel. A readily digestible wheat starch biscuit was used as a control. Ileostomy effluent was collected every 2 hours over a 16 hour period and a final collection made at 24 hours after the test meal. The monosaccharide composition and glycosyl linkages of the residual carbohydrate in the 2 hour peak period following the test meal was determined. Following consumption of the starch gel, poly- and oligo-saccharides from mucin and starch were identified in the effluent. At the peak of effluent production following the test meal, the average ratio of starch polysaccharide to mucin was 1:0.4. Of the 50 g of starch consumed, 7% of the starch escaped digestion in this fraction. Following consumption of cooked, cooled chick peas, which were fed whole or homogenized, polysaccharides deriving from starch, mucin and the cell wall were detected in the effluent. It was estimated from comparison of the composition of the food and effluent that 14% and 16% of the ingested starch in the form of homogenized and whole chick peas had escaped digestion in the small intestine. Linkage analysis showed the chemical structure of the starch escaping digestion after feeding the whole and homogenized chick peas was similar to that obtained after feeding the starch gel.     

A comparison between starch and polyacrylamide gels for the analysis of Listeria monocytogenes using multilocus enzyme electrophoresis

S.H. FLINT, N.J. HARTLEY, S.M. AVERY AND J.A. HUDSON. 1996. Forty-seven Listeria monocytogenes isolates were analysed using multilocus enzyme electrophoresis in two laboratories. Both assayed for the same six enzymes, but one used a starch gel method and the other polyacrylamide gels. The starch gel method distinguished six electrophoretic types whereas the polyacrylamide gel method produced 17 different electrophoretic types. The polyacrylamide gel method was more discriminatory than the starch gel method.     

Rheological and gelation properties of rice starch modified with 4-alpha-glucanotransferase

Rheological measurements were performed to characterize rice starch modified with 4-alpha-glucanotransferase (4alphaGTase) isolated from Thermus scotoductus, in terms of effects of the enzyme and starch concentration on flow behavior, gel strength, and melting and gelling kinetics of the modified rice starch. Consistency index decreased and flow behavior index increased with the level of enzyme treatment, and at high level of enzyme treatment, it demonstrated Bingham plastic behavior. As the level of enzyme decreased and the starch concentration increased, gelation time decreased and the final gel strength increased significantly. Regardless of treatment variables, all the modified starch gels melted at similar temperature.     

鱼类乳酸脱氢酶同工酶聚丙烯酰胺凝胶电泳技术研究

本文对用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）分离鱼类乳酸脱氢酶（ＬＤＨ）同工酶的技术进行了研究。结果表明：该方法优于淀粉凝胶电泳及琼脂糖凝胶电泳分析鱼类ＬＤＨ同工酶,具有较高的分辩率。     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

Labeling of membranes from erythrocytes and corn with fluorescamine

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.     

Chemistry and properties of starch based desiccants

Desiccants currently used in industry include molecular sieves, lithium chloride, silica gel, and corn grits. Of these, only corn grits (a form of ground corn) are biodegradable and derived from a renewable resource. A major component of the corn grits, starch, is the primary adsorptive material in the corn grits. Other polysaccharides, including cellulose and hemicellulose also have adsorptive properties. The use of alpha-amylase (EC 3.2.1.1) to modify porosity and surface properties of starch resulted in materials with enhanced water sorption properties compared to the native material. This paper reviews the chemical and structural properties of starch, corn grits, and cellulose-based scaffolds on which starch can be affixed, in order to attain structures that might someday find uses in a range of desiccant applications for industrial, commercial, and residential processes.     

On the dual role of starch,cellulose and their dialdehydes as fillers and accelerators in the tertiary amine catalysed curing of epoxy resin

Gel time studies of epoxy resins containing varying concentrations of water, starch, cellulose, or their dialdehydes were carried out at 120°C using the tertiary amines triethylamine (TEA) and hexamethylenetetramine (HEXA) as catalysts. In the 40 parts per 100 parts of resin (phr) polysaccharide-filled epoxy-HEXA system it was found that ～50% oxidised starch or cellulose produced unexpectedly high curing rate enhancements and gel times of 13 and 22 min, respectively, were obtained. With 100% oxidised starch the gel time increased to 37 min, while with 10% oxidised cellulose the gel time obtained was 91 min. The non-oxidised starch and cellulose gave even higher gel times. Thus, there seems to be some sort of a synergistic mechanism operating when the degree of oxidation of the polysaccharide is ～50% of the glucose monomer units. The difference in the effects of TEA and HEXA on the polysaccharide-filled epoxy curing reaction is explained on the basis of the decomposition of HEXA into its constituents (formaldehyde and ammonia/ammonia derivatives) under the reaction conditions employed, the formation of intermolecular and intramolecular hemiacetal and/or various hydrated aldehyde structures, and the difference in crystallinity and rigidity of the different polysaccharides affecting the availability of hydroxyl groups.     

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205 g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.     

Effects of Gamma Irradiation on Wheat Gluten

The effects of γ-irradiation on wheat gluten were studied by means of gel filtration on Sephadex G-100, starch gel electrophoresis and analysis of amino acid composition. Analyses of gluten at a moisture content of 2% revealed no significant change in amino acid composition except for cystine which was decreased by about 8% with irradiation at 10 Mrad. Changes in the chromatogram from gel filtration were interpreted in terms of random depolymerization resulting from irradiation. The results of starch gel electrophoresis suggested that irradiation levels greater than 3 Mrad resulted in characteristic changes in the molecular configuration of gliadin components.     

复合淀粉凝胶电泳同工酶分析

为了克服水解马铃薯淀粉不易获得的困难,并使“水平切片淀粉凝胶电泳同工酶分析”更容易开展,普通的化学试剂马铃薯淀粉(或精制食用马铃薯淀粉)和可溶性淀粉混合物加入适当的添加剂被用来代替水解马铃薯淀粉制作凝胶。试验结果表明：用8～10％的上述混合淀粉(5∶3),添加1％的琼脂粉和2～4％的蔗糖,所制成的“复合淀粉凝胶”可以很好地被切片,并成功地对许多不同类群的植物材料的PGM、PGI、MDH、AAT、SKDH、6PGD、IDH和ME等酶进行染色。     

Methods for starch-gel electrophoresis of sarcoplasmic proteins. An investigation of the relative mobilities of the glycolytic enzymes from the muscles of a variety of species

1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.