An Improved System for Preparative Starch Block Electrophoresis

A starch block electrophoresis system is described which includes (a) a single-unit Lucite electrophoresis chamber, (b) temperature control with a circulating water bath and casting resin-coated brass cooling plate, (c) continuous monitoring of block surface temperature and (d) an easily-assembled apparatus for rapid elution of protein from the starch segments.     

Studies on a genetically determined albumin dimer

An electrophoretic variant of human albumin has been characterized as a dimer involving both disulfide and noncovalent bonds. The variant protein was isolated by starch block electrophoresis and gel filtration and extensively investigated by gel electrophoresis and immunoelectrophoresis under a variety of conditions. No significant differences were found between normal albumin and the propositus's albumin in sulfhydryl reactivity or content or in the tryptic fingerprint.Contribution No. 140 from the Blood Research Laboratory, American National Red Cross.     

Purification and Characterization of A Human-Specific Esterase From Urine

Abstract

A human-specific esterase was isolated from urine and partially characterized by ammonium sulfate precipitation, starch block electrophoresis, and gel filtration. Its molecular weight was estimated to be 136,000 by polyacrylamide gel electrophoresis in 0.17. SDS. A diffusion coefficient of 8.6 X 10^?7 was determined by the L-plate method. Immunologic identity was shown between the urinary esterase and a human tissue esterase.     

A new convenient technique for making cell blocks

Cell block sections serve as an important diagnostic annex for cytological smears, liquid-based SurePath cytology and the Liquid-based Thin-prep Cytology Test (TCT). A variety of methods for the preparation of cell blocks are described in the literature and the techniques in cell blocks are in continuous improvement. A new technique for making cell blocks was introduced in the present study. We first used pregelatinized starch as the frame for the cell block, which is a really simple and economic method, because it can be carried out at room temperature without additional special instruments. We have performed hematoxylin and eosin (HE) staining, immunohistochemistry analysis and fluorescence in situ hybridization (FISH) in the cell block sections in 122 cytological specimens. The results demonstrated in this article show that pregelatinized starch is a useful frame for cell blocks. The pregelatinized starch can effectively collect even a few cells with powerful adhesiveness. Therefore, this new technique for making cell blocks is especially useful for cytologic samples with low cellularity, such as cerebrospinal fluid specimens.     

Collagenase production by Achromobacter iophagus.

Achromobacter iophagus produced collagenase (EC 3.4.24.3) when cultured aerobically in buffer containing 5% peptone. The bacterium is non-pathogenic and tests on rabbits indicated that the culture medium was atoxic. The collagenase, which hydrolyzed insoluble and soluble native collagen, was purified by (NH4)2 SO4 precipitation, starch block electrophoresis, and gel filtration. It was shown to be serologically distinct from Clostridium histolyticum collagenase and to have molecular weight and sedimentation coefficient values of approx. 112 000 and 5.3 S, respectively.     

Comparison of Methods for Separating Proteins Using Bisalbuminemia as a Model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and identification procedures for evaluating serum albumin variants were compared.

The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholinea prevented the complete isolation of the chicken albumin.

The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken and anti-turkey sera.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

Spontaneous dissociation of an IgG-3 paraprotein through disulfide interchange.

A paraprotein of the gamma3 subclass was observed to dissociate "spontaneously" under various experimental conditions, such as during chromatography on DEAE-cellulose and on Sephadex G-200 or on starch block electrophoresis. This phenomenon was accompanied by the formation of various complexes of higher molecular weight, displaying antigenic properties different from those of the original paraprotein. These changes did not occur in the presence of iodoacetamide, indicating that dissociation of the paraprotein was due to disulfide interchange(s) and not to the absence of interchain disulfide bridges.     

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.     

High voltage electrophoresis in starch gel of sarcoplasmatic and myofibrillar proteins of animal skeletal muscles]

The procedure for cooling starch gel during high voltage electrophoresis (voltage gradient 30-35 v/cm) of proteins of skeletal muscles of animals has been developed. In cooling the gel cuvette by ethanol of 15 divided by -20 degrees and -35 divided by -40 degrees C during electrophoresis in interrupted buffer systems of sarcoplasmatic and myofibrillar proteins the temperature of the gel plate does not exceed 26 degrees C in the most important sites. This allows heat denaturation of proteins during their separation.     

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.     

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。     

Isolation of a fibrinolytic activator from metabolites of bacillus subtilis

Summary Isolation of a crystalline fibrinolytic activator from metabolites of Bacillus subtilis was performed by salting out with ammonium sulphate and final precipitation by gradual addition of acetone. The degree of purity of the activator at the different stages of purification was tested by paper electrophoresis and starch block electrophoresis. The activator failed to attack casein, pure fibrin, gelatin and egg albumen. The reaction between the crystalline activator and plasminogen and the effect of the resulting product on fibrin were traced by paper chromatography and by recording the UV spectra of the reacting system. Finally the effects exerted by some factors on the liquefaction of the human blood clots by the activator were investigated.     

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.     

Temperature responses of dark respiration in relation to leaf sugar concentration

Changes in leaf sugar concentrations are a possible mechanism of short‐term adaptation to temperature changes, with natural fluctuations in sugar concentrations in the field expected to modify the heat sensitivity of respiration. We studied temperature‐response curves of leaf dark respiration in the temperate tree Populus tremula (L.) in relation to leaf sugar concentration (1) under natural conditions or (2) leaves with artificially enhanced sugar concentration. Temperature‐response curves were obtained by increasing the leaf temperature at a rate of 1°C min^?1. We demonstrate that respiration, similarly to chlorophyll fluorescence, has a break‐point at high temperature, where respiration starts to increase with a faster rate. The average break‐point temperature (T_RD) was 48.6 ± 0.7°C at natural sugar concentration. Pulse‐chase experiments with ¹⁴CO₂ demonstrated that substrates of respiration were derived mainly from the products of starch degradation. Starch degradation exhibited a similar temperature‐response curve as respiration with a break‐point at high temperatures. Acceleration of starch breakdown may be one of the reasons for the observed high‐temperature rise in respiration. We also demonstrate that enhanced leaf sugar concentrations or enhanced osmotic potential may protect leaf cells from heat stress, i.e. higher sugar concentrations significantly modify the temperature‐response curve of respiration, abolishing the fast increase of respiration. Sugars or enhanced osmotic potential may non‐specifically protect respiratory membranes or may block the high‐temperature increase in starch degradation and consumption in respiratory processes, thus eliminating the break‐points in temperature curves of respiration in sugar‐fed leaves.     

Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer

Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.     

Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus.

A medium was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.     

Localization and Properties of Enzymes Involved with Electron Transport Activity in Mycobacteria

A study of the subcellular localization of the nicotinamide adenine dinucleotide (NADH)-3-(4, 3-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) oxido-reductase systems in Mycobacterium was presented. Evidence based on starch gel electrophoresis and different responses of the subcellular fractions to heat inactivation suggested the existence of more than one enzyme responsible for the NADH-MTT oxido-reductase activity. One type of activity was found in the membrane-mesosome fraction which contained labile and electrophoretically non-migrating enzymes. Another type of activity was also detected in the soluble fraction which on starch gel electrophoresis exhibited 4 bands of activity, two of which showed heat resistance.     

Influence of Soy Protein Isolate Hydrolysates Obtained under High Hydrostatic Pressure on Pasting and Short-Term Retrogradation Behavior of Maize Starch

The influences of soy protein isolate hydrolysate (SPIH) obtained during different pressure treatments for 4 h on pasting and short-term retrogradation behaviors of maize starch (MS) were investigated. The results showed solubility of MS markedly increased, whereas swelling power decreased with increased SPIH concentration and pressure. Compared with native MS, the addition of SPIH led to decrease of peak viscosity, final viscosity, setback, and breakdown, whereas pasting temperature was increased. Meanwhile, differential scanning calorimetry (DSC) analysis also showed an increase in gelatinization temperature. In addition, low-field nuclear magnetic resonance (LF-NMR) analysis indicated that the tight association of water and starch molecules was observed with increasing pressures and additions of SPIH. Confocal laser scanning microscopy (CLSM) and atomic force microscope (AFM) images indicated that SPIH obtained at 200 MPa dispersed in the MS gel system to block the formation of hydrogen bonds and inhibit the recrystallization of MS. Fourier transform infrared (FTIR) spectroscopy analysis demonstrated that the addition of SPIH resulted in a decrease in hydrogen bonds within the starch molecules and the result supported above CLSM and AFM measurement results. The results proved that the addition of SPIH could effectively influence pasting characteristics and inhibit the short-term retrogradation of MS, which can be helpful to the application of SPIH in starch-based functional foods.

     

Thermal sensitivity of NAD-malate dehydrogenase isoforms from various Pennisetum ecotypes

Thermal sensitivity of NAD-malate dehydrogenase (NAD-MDH) was studied in several Pennisetum ecotypes. The effect of temperature was investigated using either crude extracts from shoots or separated isoforms. In all the ecotypes tested, in vitro temperature optima were found to be near 45°C. A high heat stability of NAD-MDH was observed for all the ecotypes studied. Three isoenzymes were isolated by DEAE-cellulose chromatography and analyzed by starch gel electrophoresis. Three anodic isoenzymes were separated on the electrophoretic pattern, they are known to be associated with peroxisomes, mitochondria and soluble leaf compartments, respectively. The soluble NAD-MDH isoenzyme was found to be more affected at high temperature than the peroxisomal and mitochondrial forms.