Electrophoretic Characteristics of Staphylococcal Hyaluronate Lyase

Partially purified staphylococcal hyaluronidase was studied with respect to its electrophoretic properties by the use of starch slurry or semisolid Noble agar as a matrix. The polarity of enzyme activity was found to be cathodal on starch and anodal on agar gel. The electrophoretic migration of the partially purified enzyme on starch, as a function of pH, suggested that the enzyme has an isoelectric point of pH 9.5 to 10.     

New insights in agar biorefinery with arylsulphatase activities

Agar is a major gelling agent used both in food and pharmaceutical applications. Traditional purification of agar is generally performed by sequential time consuming chemical and/or physical steps, leading to both poor recovery yields and low productivities. As a consequence, only 30% of the amount of agar produced is actually available under purified form to feed the world market.The current limiting factor for purification is the presence of sulphated compounds such as sulphated-agaropectin, which strongly affect the technological properties of the agar gel such as gel strength, melting and fusion temperatures and electroendosmosis.In this context, this communication aims at discussing about the development of a biorefining agar purification approach which allows overcoming the current limitations associated with traditional purification methods. More specifically, this article focuses on the potential role of arylsulphatases in agar purification processes to reduce the number of purification steps and to improve recovery yields.This review first presents the global gelling agents market before focusing on agar characteristics and production processes. Then, after a brief reminder of the sulphur metabolism, the roles, classes and properties of the different arylsulphatases are described to draw perspectives on their integration in current or new agar production processes.     

Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin.

Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.     

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Abstract Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.     

Failure to detect mouse species-specific antigens in Toxoplasmin Sevac.

Toxoplasmin--a highly purified extract from the protozoa Toxoplasma gondii propagated in mice--was tested for the presence of the mouse species-specific antigens by immunodiffusion in agar gel and by PCA test. Neither test gave positive reaction. It was concluded therefore that Toxoplasmin Sevac used for the detection of dermal hypersensitivity to toxoplasma antigens in humans does not contain any detectable contamination by mouse species-specific antigens within the limits of sensitivity of both methods used.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Heterologous expression and characterization of a recombinant thermophilic arylsulfatase from Thermotoga maritima

Production of low sulfated agar or agarose from agar or agaropectins by enzymatic hydrolysis has advantages but a high melting temperature is needed. The arylsulfatase gene from thermophilic Thermotoga maritima was cloned and expressed in Escherichia coli W3110 with pCol-MICT as the vector. The gene was comprised of 1,782 bp and encoded a protein of 593 amino acids with a molecular weight of 65 kDa. The recombinant arylsulfatase was partially purified by heat treatment (70°C, 30 min) and characterized. The enzyme was prepared with a total protein content of 2.4 mg and a specific activity of 20.63 U/mg. Optimal temperature and pH of the enzyme were 80°C and 7.0, respectively, for hydrolysis of p-nitrophenyl sulfate and sulfate content of agar was diminished to 40% after a 12 h treatment at that condition. Enhanced electrophoretic movement of DNA was observed in enzymetreated agar gel compared to that in a non-treated agar gel. These results suggest that thermophilic arylsulfatase expressed in E. coli could be useful for producing a low sulfated agar and electrophoretic grade agarose.     

Extraction of nontype-specific antigens of group A Streptococcus by potassium thiocyanate

A method for extraction of nontype-specific antigens of the group A streptococcus cellular wall from whole microbial cells is described. Potassium thiocyanate was used as an extracting agent. Nontype-specific antigens of the thiocyanate extract purified by ammonium sulfate precipitation were examined by immunodiffusion in agar gel. The thiocyanate extracts were found to contain several nontype-specific protein antigens part of which were absent from the HCl-extracts. No group A streptococcal polysaccharide was found in the thiocyanate extracts.     

Identification of a Marine Agarolytic Pseudoalteromonas Isolate and Characterization of Its Extracellular Agarase

The phenotypic and agarolytic features of an unidentified marine bacteria that was isolated from the southern Pacific coast was investigated. The strain was gram negative, obligately aerobic, and polarly flagellated. On the basis of several phenotypic characters and a phylogenetic analysis of the genes coding for the 16S rRNA, this strain was identified as Pseudoalteromonas antarctica strain N-1. In solid agar, this isolate produced a diffusible agarase that caused agar softening around the colonies. An extracellular agarase was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on DEAE-cellulose. The purified protein was determined to be homogeneous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it had a molecular mass of 33 kDa. The enzyme hydrolyzed the β-1,4-glycosydic linkages of agar, yielding neoagarotetraose and neoagarohexaose as the main products, and exhibited maximal activity at pH 7. The enzyme was stable at temperatures up to 30°C, and its activity was not affected by salt concentrations up to 0.5 M NaCl.     

Sporulation promoting factor in vegetative cells of Bacillus subtilis

A simple experimental system for detection of sporulation promoting factors was presented. This system showed that there was a sporulation promoting factor in the vegetative cells of Bacillus subtilis cultivated on nutrient agar for 9 hr (at stage T0). The factor was partially purified from the sonicate of vegetative cells by ethanol fractionation, gel filtration, chromatography and preparative gel electrophoresis, and it was identified as manganese-containing protein.     

Purification of Clostridium botulinum Type F Progenitor Toxin

Clostridium botulinum type F progenitor toxin was purified to a homogeneous state as judged by gel filtration on Sephadex G-200, ultracentrifugation, and disc electrophoresis. The sedimentation constant, corrected to water at 20 C, of type F progenitor toxin was determined to be 10.3 and the molecular weight to be 235,000 by ultracentrifugation at pH 6.0. The purified toxin contained a toxicity of 1.2 x 10(8) 50% lethal doses/mg of N. In agar gel double diffusion, it formed two precipitin lines at pH 6.0. The progenitor toxin of type F differs from that of type A in that it contains no hemagglutinin and from that of type E in that it is not activable.     

Episome-carried Surface Antigen K88 of Escherichia coli II. Isolation and Chemical Analysis

The K88 antigen was carried by episomal transfer to D282, a nonmotile Escherichia coli strain without K antigen. D520, obtained by this episomal transfer, was used for the extraction of K88 antigen. It was shown by the agar gel precipitation technique that some K88 antigen was released from D520 into suspending aqueous medium. The amount of liberated material was increased by gentle heating (60 C) or treatment in a Waring Blendor. The antigen was obtained from the extracts in a purified form by making use of its insolubility between pH 3.5 and 5.5 and of its high sedimentation rate (S(0) (20,w) = 36.7S). The homogeneity of the material was demonstrated by agar gel precipitation with D520 antiserum, by analytical ultracentrifugation, and by moving-boundary electrophoresis. Chemical analysis revealed that K88 is a pure protein containing all the common amino acids with the exception of cysteine-cystine. Purified K88 selectively precipitated the K88 antibodies from D520 antiserum and was shown to be immunogenic in rabbits.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.     

Steroid receptors in the human prostate. 1. Estradiol - 17beta binding in benign prostatic hypertrophy.

Agar gel electrophoresis and ultracentrifugation on continuous sucrose gradients revealed the presence of a 4S estradiol 'receptor' in cytosols of samples of human benign hyperplastic prostate tissue. High affinity (charcoal stability) and saturability (disappearance with excess competitor) binding characteristics of the molecular species concerned facilitated its clear distinction from similarly migrating serum albumin-steroid complexes. Our data, obtained with human serum, purified human albumin and albumin-enriched cytosol strongly suggest that agar gel electrophoresis, when used alone, may lack specificity for the quantification of estrogen 'receptors'. Radioligand binding to these molecules may be obscured by similarly migrating albumin-steroid complexes which fail to dissociate during electrophoresis. We advocate the inclusion of competitor experiments to improve the specificity of the method.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

Substance immunologically cross-reactive with insulin from murine myeloid leukemia: purification and characterization

A substance immunologically cross-reactive with insulin (SICRI) appears in murine myeloid leukemia. Progression of disease is paralleled by the increase of SICRI levels in the serum; this increase of SICRI levels did not correlate with a decreased concentration of circulating glucose. SICRI was isolated and purified from spleen infiltrated with leukemic cells. Monospecific antiinsulin immunoglobulin G was used for immunoaffinity chromatography to isolate SICRI from tumor tissue. The purified substance yielded a single band with a molecular mass of about 150 kDa in polyacrylamide gel electrophoresis under denaturating and non-denaturating conditions. Purified SICRI enhanced growth of myeloid leukemia cells in soft agar. Biochemical and biological data together with our previous results obtained in other experimental tumors provide evidence that SICRI and insulin are two distinct biologically active agents. SICRI plays a role in murine myeloid leukemia as an autocrine growth promoting factor.     

血红蛋白酸性琼脂电泳及其临床应用

本文介绍一种酸性琼脂电泳方法。它可以比较容易地分开血红蛋白A和血红蛋白F、可将异常血红蛋白分成两大类,即酸性电泳阳性和酸性电泳阴性两类异常血红蛋白。此法在血红蛋白病中比较常用的是鉴别血红蛋白S与其它电泳速度相同的变异物,帮助诊断镰状细胞贫血。在常见病方面,这种方法还能分开血红蛋白A和糖基化血红蛋白,用来帮助诊断糖尿病。     

Purification and Properties of an Extracellular Agarase from Alteromonas sp. Strain C-1

A marine bacterial strain isolated from the Bay of San Vicente, Chile, was identified as Alteromonas sp. strain C-1. In the presence of agar, this strain produced high levels of an extracellular agarase. The production of agarase was repressed by glucose, with a parallel decrease in bacterial growth. The enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with an overall yield of 45%. The enzyme has a molecular weight of 52,000, is salt sensitive, and hydrolyzes agar, yielding neoagarotetraose as the main product, with an optimum pH of about 6.5.