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At least 19 recordsLinked to original sources
PubMed1975

Microheterogeneity of Immunoglobulin G from plasmocytomas. Identification of two types of IgG by isoelectric focusing.

Isoelectric focusing of serum immunoglobulin G (IgG) revealed that the microheterogeneity, which is expressed in the isoelectric points (IEP), partly is caused by differences in the content of N-acetyl-neuraminic acid (NANA), partly by other effects, probably including deamidation. Two different types of Plasmocytoma-IgG, which differ in IEF-pattern, i.e. population distribution, in sensitivity towards neuraminidase and in carbohydrate content, are described. The contents of carbohydrates in these 2 IgG-types are up to 30% greater and 50% less, respectively, than that of normal IgG. A precise correlation was found between the total content of NANA in moles per mole IgG-monomer and the shift in band-IEP on hydrolysis catalyzed by neuraminidase. This may be used for a rapid estimate of NANA in IgG. The results do not permit a discrimination between an anabolic and a catabolic origin of the heterogeneity in the carbohydrate moiety of IgG.

Antibodies, Heterophile↗
PubMed1978

Influence of fatty acid and time of focusing on the isoelectric focusing of human plasma albumin.

In the isoelectric focusing of human plasma albumin, two major peaks of pI 4.8 and 5.6 are observed. As fatty acids are removed from the albumin either by defatting before the focusing experiment or gradually during the focusing experiment, the pI 4.8 peak diminishes and the pI 5.6 peak increases. The interpretation of this effect is confirmed by fatty acid analysis before and after focusing. Alkylation of the free sulfhydryl group changes the focusing position of the defatted peak to pI 5.7. Otherwise the isoelectric focusing pattern of human albumin is unaffected by the status of the free sulfhydryl group or by the ampholine concentration.

Alkylation↗
PubMed1975

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.

Gels↗
PubMed1978

Fractionation of proteins from Rous sarcoma virus and avian myeloblastosis virus by isoelectric focusing.

The isoelectric focusing technique in the pH gradients was used for a preparative isolation of proteins from Rous sarcoma virus and avian myeloblastosis virus. The purified major gs protein, p27 (pI = 9.1) and the gP86 (pI = 5.3) were obtained after disruption of virus with 1% non-ionogenic detergent in the presence of 6M urea. The p10, p15, and p19 were present in the same range of pH (pI = 6.8). A strongly basic protein, immunologically active, presumably the p12, was found in the alkaline region of the pH gradient 10.8. These proteins fully retained their immunological activity. On the other hand, in the acidic region of the pH gradient between pH 4 and 5, strong precipitates were regularly found. These precipitates were complexes which were formed by interaction of the acid components of ampholines with the viral proteins during isoelectric focusing. Almost all viral proteins were present, differing only in quantity. The complexes were stabile in 1% non-inogenic detergent and 6M urea. They were dissociated with 1% SDS and 5M urea, and had no immunological activity. The methods of virus disruption and possibilities of formation of the ampholine-viral protein complexes are discussed.

Avian Leukosis Virus↗
PubMed1977

A comparative study of haemoglobins from the Artiodactyla by isoelectric focusing.

1. Isoelectric focusing on 7% acrylamide gels in 2% ampholyte of pH range 6-8 was used to fractionate the haemoglobins of 81 animals, representing 30 species from 4 families of the Order Artiodactyla. 2. Isoelectric points of the major haemoglobins were determined and the relative distribution of minor haemoglobins compared. 3. Marked haemoglobin heterogeneity was observed, with polymorphic and multiple haemoglobins. No one haemoglobin was common to all species.

Animals↗
PubMed1975

Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

SS-interchanged and oxidized isomers of bovine serum albumin separated by isoelectric focusing.

Column isoelectric focusing separates commercial bovine serum albumin in 5 fractions with isoionic points in the vicinity of that of mercaptalbumin (pI 5.24). About 20% of the bovine albumin have isoionic points higher than mercaptalbumin and are split into two fractions, both recognized as SS-interchanges isomers: (1) pI 5.39 is the "aged" albumin described by Nikkel and Foster (1971, Biochemistry 10, 4479); (2) pI 5.45 represents a further degree of SS-interchange, catalyzed by small amounts of cysteine in the solution ('cysteine-aged' albumin). In 6 M urea the "cysteine-aged" albumin is electrofocused to the same pH value as mercaptalbumin. In 6 M urea 40% of commercial albumin focuses in 3 fractions with isoionic points lower than mercaptalbumin. This percentage will increase during incubation at oxidizing conditions ("oxidized" albumin). Electrofocused in water the oxidized fractions have isoionic points at pI 5.28, 5.18 and 5.12, respectively. The shifts in isoionic point of the "oxidized" albumins are caused by irreversible changes in the primary structure. Although the free SH group of albumin is oxidized during the oxidation reaction, the observed changes in isoionic points are caused by modifications of some other amino acid residues. Both "cysteine-ageing" and "oxidation" are inhibited by alkylation of the SH group. "Cysteine-ageing" is furthermore inhibited when the bovine albumin is "oxidized".

Animals↗
PubMed1979

Improved identification of antitrypsin phenotypes through isoelectric focusing with dithioerythritol.

Isoelectric focusing has replaced acid starch-gel electrophoresis for the routine determination of antitrypsin phenotypes in recent years. We observed increased sharpness of antitrypsin bands and decreased background stain following the addition of DTE to serum before isoelectric ofcusing in polyacrylamide gels. DTE and DTT were the only sulfhydryl reducing agents which produced this effect. Electrophoretic mobility of antitrypsin bands was increased very slightly. Decreased background stain resulted from the precipitation of albumin. Precipitation (coagulation) of albumin was complete in serum after 60 min incubation at 37 degrees C at concentrations of 30 mM DTE or DTT. Optimal pH for denaturation was 7.6 to 8.8. Ionic concentrations reduced the strength of the coagulum at 2.5M sodium chloride but had little effect at lower concentrations. Marked temperature effects were noted. As a result of these studies, we recommend examination of native and reduced (30 mM DTE) serum on isoelectric focusing in polyacrylamide gel for all samples submitted for routine antitrypsin phenotype determinations. It also seems possible that the nontoxic DTE (DTT) precipitation of albumin could prove useful for studies of serum proteins other than alpha 1-antitrypsin.

Animals↗
PubMed1979

Characterization of free and tightly bound lipoprotein in intima by thin layer isoelectric focusing.

Thin layer isoelectric focusing followed by second dimension quantitative immunoelectrophoresis has been used to characterize the free and tightly bound lipoprotein (lp) fractions in human aortic intima. In 3.3% acrylamide gels plasma low density lipoprotein (LDL) focuses rapidly, but very low density lipoprotein (VLDL) fails to enter the gell and remains at the origin (point of application). Samples of normal intima and lesions were placed directly on the gel and focused for 10--12 h; in normal intima and gelatinous lesions which had not accumulated cholesterol 60--70% of the free LP focused with plasma LDL, but with increasing accumulation of cholesterol the proportion focusing with LDL decreased and the component remaining at the origin increased. In 9 of the 39 samples examined one or more components with pI acid to LDL were present, and in one sample the whole peak focused at a more acid pI. In 4 of 5 lipid-rich centres of gelatinous plaques all the free LP remained at the origin, and the bound LP released by incubation with plasmin remained at the origin in all samples in 3.3% gels although it would focus in 2.6% gels. With antiserum to apo-C this large LP did not behave like VLDL, and it appears to be an aggregated LDL. An increased proportion of aggregated LDL was associated with (a) accumulation of cholesterol, (b) topographical location towards the centre of plaques, and (c) partial destruction of endogenous LP during incubation of fresh samples of tissue.

Acrylamides↗
PubMed1978

Estradiol receptor analysis in human breast cancer tissue by isoelectric focusing in polyacrylamide gel.

Isoelectric focusing in polyacrylamide gel combined with limited proteolysis is a simple and specific method for quantitation of estradiol receptors in breast cancer tissue. At least eight different samples can be analyzed simultaneously on one gel, and the whole procedure, including sample preparation, takes less than 7 hr. In comparison with sucrose gradient centrifugation, isoelectric focusing is more sensitive, possibly due to the short time (1.5 to 2 hr) needed for the analysis. Furthermore, only one incubation with tritium-labeled estradiol is needed for an analysis, which means that a smaller amount of tumor tissue is needed than for most other methods. This fact allows analysis of the estrogen receptor content in tumor material obtained from fine-needle biopsy.

Breast Neoplasms↗
PubMed1979

Resolution of products of the duplicated hemoglobin alpha-chain loci by isoelectric focusing.

Using a high-resolution isoelectric focusing system, we have been able to separate the products of the individual "nonallelic+ mouse hemoglobin alpha-chain genes. The hemoglobins of mice of inbred strains that make two structurally different, but electrophoretically identical, alpha chains resolve into two bands on isoelectric focusing. The hemoglobin from other strains that make only one type of alpha chain forms a single band. Two new "alleles" of haplotypes of Hba (the alpha-chain complex locus) are described that code for hemoglobins with solubilities similar to that of the hemoglobin of strain C57BL/6J, but that give demonstrably different isoelectric focusing patterns. Because this method allows Hba typing of strains with the Hbbd or "diffuse" allele at the hemoglobin beta-chain locus, we have also been able to classify a number of previously untyped stocks, including some recently derived from feral populations. Among these mice we have found new representatives of most of the extant Hba genotypes. The demonstration that it is possible to detect genetic differences involving only neutral amino acid substitutions between proteins suggest that careful application of appropriate isoelectric focusing systems to the analysis of other proteins will greatly increase the sensitivity of our ability to detect genetic variation.

Amino Acid Sequence↗
PubMed1978

Methods of isoelectric focusing.

The method of isoelectric focusing has been avoided by many workers because of expense, technical difficulty, and problems of interpretation. Inexpensive, easy, and interpretable results are possible using equipment and reagents commonly available. Methods which allow these results are presented and explained.

Isoelectric Focusing↗
PubMedDate not supplied

[Study of the yeast dissolving enzymic complex by isoelectric focusing].

By isoelectric focussing the lyzing complex produced by Actinomyces griseinus-11 has been fractionated. The sole neutral protease which is active at pH 7.0 does not participate in lysis induced by other enzymes. The lyzing activity of this complex is associated with the carbohydrase enzymes that are at least three in number. Various carbohydrases may exert a synergistic effect upon their combined action on the protein-vitamin concentrate.

Actinomyces↗
PubMed1978

Multiple molecular forms of certain lysosomal enzymes separated by isoelectric focusing on cellulose acetate membranes.

Analytical isoelectric focusing on cellulose acetate membranes offers greater resolution of the enzyme pattern than does standard electrophoresis and offers numerous advantages over other support media in isoelectric focusing. A series of lysosomal enzymes, deficiencies of which cause lethal storage diseases in infants and children, can be shown to exist as multiple molecular forms by analytical isoelectrical focusing on cellulose acetate membranes. This analytical technique should be a valuable tool in the investigation of enzyme polymorphism and genetically determined enzyme deficiency diseases.

Acetates↗
PubMed1979

Direct tissue isoelectric focusing in agarose.

We are isoelectric focusing small amounts of tissues placed directly on electroendosmosis-free agarose gels instead of using the saline extracts of tissue homogenates. More numerous proteins are extracted during isoelectric focusing of the solid tissues than present in the same tissues.

Adenocarcinoma↗
PubMed1977

[Microheterogeneity of R plasmid coded beta-lactamases in analytical isoelectric focusing (author's transl)].

Analytical isoelectric focusing of plasmid-coded beta-lactamases in polyacrylamide gels gives rise to several satellite bands in addition to one major active band (microheterogeneity). When the R plasmids are transferred by conjugation into 4 different bacterial species (Escherichia coli, Klebsiella pneumoniae, Salmonella typhi-murium, Proteus mirabilis) the position of the main band remains constant, but the number and position of the satellite bands are changed. Furthermore, the minimal inhibitory concentrations of several beta-lactamines are also changed when the plasmids are transferred between these different host strains. These facts suggest that the bacterial host might modify the physical structure and the activity of plasmid-determined beta-lactamases. They also suggest that identification of beta-lactamases and comparison of their isoelectric focusing patterns should always be carried out using the same bacterial host strain.

Amidohydrolases↗
PubMed1975

Human and monkey prolactin and growth hormone: separation of polymorphic forms by isoelectric focusing.

The feasibility of using isoelectric focusing for the separation of primate pituitary growth hormone from prolactin and for the characterization of polymorphic forms of these hormones was explored. In a pH 3--10 gradient, extracts of both human and cynomolgus monkey pituitaries were each resolved into 4 growth hormone components and at least 3 prolactin components, as shown by radioimmunoassay. In narrower gradients (of 2--3 pH units) greater resolution was achieved; the principal growth hormone components were well separated from the principal prolactin components but there was overlapping of some minor components. A partially purified human prolactin preparation was found to contain 4 prolactin components, one of which had a prolactin/growth hormone ratio of 760. Clinical grade human growth hormone was also resolved into at least 5 prolactin and 5 growth hormone components, many of which had higher pI values than those found in pituitary extract. Under the conditions used, both growth hormone and prolactin were found to be polymorphic with respect to isoelectric point. Some of the human prolactin components were found to contain less than 0.2% growth hormone by radioimmunoassay. Monkey growth hormone containing 0.01% prolactin was isolated. These findings demonstrate that isoelectric focusing is useful for the preparation of both growth hormone and prolactin which are essentially free of one another. Furthermore, the polymorphic forms were repeatedly found in preparations obtained by several methods and from 2 different species, suggesting that these forms are not artifacts.

Animals↗