Thin-layer Isoelectric Focusing Research Articles

Isoelectric-focusing of galactokinase in lens and other tissues

Thin-layer isoelectric focusing was used to investigate galactokinase in lenses from humans, cows, rats, rabbits and also in other tissues (red cells, liver, kidney, brain, placenta) from these species. In each case the enzyme activity was present as a single isozyme. The isoelectric point was the same in lens, red cell and other tissues of the same species, but differed from species to species. Post-translational modification due to deamidation was not detected in the lens or red cell from these species. Galactokinase activities in whole tissue extract were determined in different tissues of the various species. There were wide variations in activity. In the adult human lens, both normal and cataractous, it was low. The findings indicate that isoelectric focusing of RBC galactokinase combined with specific enzyme staining allows one to monitor lens galactokinase in patients with cataracts.

Serum creatine kinase MM sub-band determination by isoelectric focusing: a potential method for the monitoring of myocardial infarction

Fresh myocardium homogenates analyzed by thin-layer isoelectric focusing revealed the presence of two prominent creatine kinase (CK; EC 2.7.3.2) sub-bands, MMO (pI 7.10) and MM1 (pI 6.88), in approximately equal proportion. While these forms represented together as much as 85% of the cellular MM fraction, they accounted only for viz. 2.2 and 27.7% of the total serum MM activity when measured 8 h before the CK peak in patients with myocardial infarction. Incubation of the isolated MMO and MM1 with normal human serum demonstrated that the former turned to MM1 within 5 h at 37°C; further changes affecting MM1 gave rise to other sub-bands, MM2 (pI 6.70), MM3 (pI 6.45), and MM4 (pI 6.25). In our patient population, these three forms represented more than 75% of the serum CK-MM activity at the CK peak; hence, soon after the enzyme release, the serum MM isoenzyme mainly consists of degradation products arising from the labile MMO and MMl. Among the two cellular forms, MMO was the best related to the total enzyme activities and the most efficient for differentiating the patients with left ventricular failure from the others during the entire survey period ( F = 3.8, p < 0.05). Because its presence in the blood provides evidence for a very recent CK release from the tissues, serum CK-MMO determinations might be proposed for following the extension of the lesion after a myocardial infarct.

Rapid differentiation of Artemia spp. Populations by thin layer isoelectric focusing

Artemia spp. of various geographic origins, yearly collection lots, developmental stages, and a binary geographic mixture were analyzed by thin layer polyacrylamide gel isoelectric focusing (IEF). IEF of geographical Artemia samples yielded unique profiles for each population in the pH range employed. Parthenogenetic populations of Artemia exhibited IEF patterns which were, generally, dominated by fewer proteins; bisexual populations exhibited more evenly distributed, complex protein patterns. There were similarities reflected in IEF profiles of geographically isolated Artemia descended from a common ancestry. Yearly collections of Artemia exhibited no apparent differences in IEF protein patterns. There were differences noted in three developmental stages of Artemia. A binary mixture of geographic populations yielded IEF patterns intermediate to those of the component pure samples.

Purification and characterization of component A of the methane monooxygenase from Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) possesses a multi-component methane monooxygenase which catalyzes in vivo the conversion of methane to methanol. Component A of this enzyme system, believed to be the oxygenase component, has been purified to near homogeneity (95%). The native protein has a molecular weight of approximately 210,000 and is comprised of three subunits of Mr = 54,000, 42,000, and 17,000, which appear to be present in stoichiometric amounts suggesting an alpha 2, beta 2, gamma 2 arrangement in the native protein. Purified preparations of the protein are virtually colorless and examination of the uv/visible absorption spectrum revealed a peak around 280-290 nm and thereafter a steady decrease in absorbance to longer wavelengths. The ESR spectrum of the oxidized protein gave a signal at g = 4.3, presumably due to rhombic iron, and a radical signal at g = 2.01. Upon reduction with dithionite, a signal at g = 1.934 appeared. Chemical analyses of our purified preparations revealed the presence of iron (2.3 mol/mol) and zinc (0.2-0.5 mol/mol): molybdenum, copper, nickel, heme, and acid-labile sulfur were all virtually absent. On ultra thin layer isoelectric focusing, purified component A was judged to have a pI between 5.0 and 5.1. Extracts prepared from a variety of other methanotrophs failed to show any cross-reaction to antibody raised against M. capsulatus component A.

Low protein variability and genetic similarity between populations of the polar bear (Ursus maritimus)

Blood samples from a total of 460 polar bears (Ursus maritimus) from various Arctic regions, but excluding the USSR, were collected during the period 1967-1981 to study electrophoretic variation in different proteins. Two hundred and one samples from Alaska, 48 from the Canadian Arctic, 89 from Svalbard, and 21 from Northeast Greenland were collected during the period 1967-1973 and were analysed by vertical polyacrylamide gel electrophoresis to study transferrin and hemoglobin polymorphism, Thirty-one samples collected in 1974 were analysed by starch gel electrophoresis for 14 enzyme systems in serum and red blood cells. Seventy samples collected from Alaska, the Barents Sea, and Canada in 198&81 were studied by starch gel electrophoresis, and further analysed for protein variation by thin-layer isoelectric focusing, horizontal polyacrylamide gel electrophoresis, and two-dimensional electrophoresis. In all, about 75 loci were analysed for variation. The degree of protein and enzyme variation in the polar bear was observed to be relatively low. Starch gel electrophoresis revealed variation of an unidentified serum protein. The distribution of this protein indicates a closer connection between bears in Alaska and Canada compared to those in Greenland and Svalbard, but the differences were not significant. As in many large mammals, the information from protein variation in polar bears has limited use for management purposes. We could not find any simple system usable for identification of discrete populations. On the basis of protein variation as sole criterion, the populations investigated could not be separated. Possible explanations for the uniformity of blood proteins can be exchange of bears between geographical areas and/or a high selective pressure in polar bears.

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases.

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.

Tissue distribution of mammalian aldose reductase and related enzymes

Activities of aldose reductase (AR) and related NADPH-dependent enzymes were examined in extracts of human, cat, dog, guinea pig, mouse, monkey, pig, rabbit, rat and sheep lenses and a variety of other tissues. The activity of the tissues against dl-glyceraldehyde, d-glucuronic acid, and 3-pyridinecarboxaldehyde (PCA) was determined. High glyceraldehyde:glucuronic acid activity ratios, a characteristic of aldose reductase, were found in all lenses, except from mouse. An analytical thin-layer isoelectric focusing system which separates the mammalian NADPH-dependent enzymes was developed. AR appears to be present as two or more isozymes in all mammalian lenses studied with the exception of mouse. Other tissues contain one or more isozymes which have the same isoelectric point and substrate specificity as the AR present in the lens of that species. This AR activity, however, may represent only a small proportion of the total NADPH reducing activity present. AR and HDH isozymes reduce the aromatic substrate, PCA, and thus have the general characteristics of an aldehyde reductase.

Genetics of four plasma protein loci in Equus przewalskii: new alleles at the prealbumin, postalbumin and transferrin loci.

This paper reports genetic variation at the prealbumin (Pr), postalbumin (Pa) and transferrin (Tf) loci in Equus przewalskii found using thin layer isoelectric focusing and an amphoteric separator. The method resolves all three loci plus serum esterase (Es) on a single gel, and typing of all four loci is readily achieved. In addition to the esterase alleles previously reported by Fisher & Scott (1979), five alleles were found at the Pr locus, three at the Pa locus and six at the Tf locus. Analysis of several mating types confirms inheritance is autosomal and codominant for all four loci.

Low protein variability and genetic similarity between populations of the polar bear (Ursus maritimus)

Blood samples from a total of 460 polar bears (Ursus maritimus) from various Arctic regions, but excluding the USSR, were collected during the period 1967-1981 to study electrophoretic variation in different proteins. Two hundred and one samples from Alaska, 48 from the Canadian Arctic, 89 from Svalbard, and 21 from Northeast Greenland were collected during the period 1967-1973 and were analysed by vertical polyacrylamide gel electrophoresis to study transferrin and hemoglobin polymorphism, Thirty-one samples collected in 1974 were analysed by starch gel electrophoresis for 14 enzyme systems in serum and red blood cells. Seventy samples collected from Alaska, the Barents Sea, and Canada in 198&amp;81 were studied by starch gel electrophoresis, and further analysed for protein variation by thin-layer isoelectric focusing, horizontal polyacrylamide gel electrophoresis, and two-dimensional electrophoresis. In all, about 75 loci were analysed for variation. The degree of protein and enzyme variation in the polar bear was observed to be relatively low. Starch gel electrophoresis revealed variation of an unidentified serum protein. The distribution of this protein indicates a closer connection between bears in Alaska and Canada compared to those in Greenland and Svalbard, but the differences were not significant. As in many large mammals, the information from protein variation in polar bears has limited use for management purposes. We could not find any simple system usable for identification of discrete populations. On the basis of protein variation as sole criterion, the populations investigated could not be separated. Possible explanations for the uniformity of blood proteins can be exchange of bears between geographical areas and/or a high selective pressure in polar bears.

β- d-mannosidase from Helix pomatia

β- d-Mannosidase (β- d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β- d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α- d-mannosidase, β- d-galactosidase, 2-acet-amido-2-deoxy- d-glucosidase, (1→4)-β- d-mannanase, and (1→4)-β- d-glucanase activities, almost devoid of α- d-galactosidase activity, and contaminated with <0.02% of β- d-glucosidase activity. The purified enzyme had the same K m for borohydride-reduced β- d-manno-oligosaccharides of d.p. 3–5 (12.5m m). The initial rate of hydrolysis of (1→4)-linked β- d-manno-oligosaccharides of d.p. 2–5 and of reduced β- d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β- d-mannopyranosides were readily hydrolysed. β- d-Mannobiose was hydrolysed at a rate ∼25 times that of 6 1-α- d-galactosyl-β- d-mannobiose and 6 3-α- d-galactosyl-β- d-mannotetraose, and at ∼90 times the rate for β- d-mannobi-itol.

Biochemistry of the ageing rat lens. II. Isoelectric focusing of water-soluble crystallins.

Thin-layer isoelectric focusing of the water-soluble crystallins shows with increasing age of the animals an increase of alpha-crystallins of high and low molecular weight and of the total beta-crystallins content. The beta-crystallin components of high molecular weight also increased during ageing, but the beta-crystallins of lower molecular weight decreased considerably. The total gamma-crystallin content, as well as certain gamma-crystallin components also decreased considerably with increasing age, or vanished completely. From 429 to 1,211 days of age the male gamma-crystallins as percent of the water-soluble moiety, were higher than the female gamma-crystallins. As a consequence of ascending age, the content of water-insoluble proteins of the rat lens increased gradually and continuously, e.g. by a process of insolubilization of soluble beta- and gamma-crystallins.

Identification of species specific hemoglobin by isoelectric focusing

A method for the discrimination of human hemoglobin from animal sources is described. Hemoglobins from 34 species with and without treatment by p-chloromercuri-benzoate (PCMB) were examined by thin-layer isoelectric focusing (IEF) in polyacrylamide gel (PAG) plates containing an Ampholine gradient of pH 3.5 – 9.5. PCMB treatment was effective in distinguishing human and animal hemoglobins with their characteristic IEF patterns. Values for the isoelectric points of the major and minor hemoglobins of each species are also described.

Neonatal screening of β‐thalassemias by thin layer isoelectric focusing

As an alternative to the recently proposed screening for beta-thalassemias by isoelectric focusing (IEF) of denatured globin chains in urea-detergent gels [6], IEF of intact hemoglobins, obtained from umbilical cord blood in neonatal units, is reported here. For maximum separation, IEF should be performed in nonlinear pH gradients, in gels containing 0.2 M beta-alanine and 0.2 M 6-amino caproic acid, which flatten the pH gradient around pH 7, thus increasing the resolution between HbA and HbFac. The method can unambiguously detect homozygous and heterozygous beta-thalassemic conditions. A bimodal distribution of HbA at birth has been found: In heterozygous patients, HbA values of 9.02% (range 6.8-9.98%) have been found while in normal newborns HbA levels of 20.34% (range 11.02-30.6%) have been demonstrated.

Fatty acid binding protein. Isolation from rat liver, characterization, and immunochemical quantification.

Fatty acid-binding protein (FABP) was identified and isolated from rat liver cytosol by gel filtration, thin layer isoelectric focusing, and affinity chromatography. FABP (Mr 12,080 +/- 80) exists in several immunochemically identical forms differing in isoelectric pH, which may in part reflect differences in their respective complements of bound endogenous ligand. FABP-bound fatty acids accounted for 60% of total cytosolic long chain fatty acids but contained no detectable phospholipid; the substantial enrichment of FABP in 18:2 and 20:4 as compared with whole liver homogenate was not influenced by homogenization of tissue in EDTA. The amino acid composition of FABP suggests that it is closely related or identical with certain similar neutral and acidic cytosolic proteins reported from other laboratories. By quantitative radial immunodiffusion, FABP concentration in cytosol from livers of sexually mature female rats exceeded that from mature males (51.7 +/- 3.0 versus 39.8 +/- 4.0 micrograms/mg of protein, p less than 0.05), confirming earlier studies in which sex steroid effects on rates of fatty acid utilization were correlated with FABP concentration as determined by means of a binding assay. The abundance of FABP, its importance in the cytosolic binding of endogenous as well as exogenous fatty acids, and its demonstrated correlation with rates of hepatocyte fatty acid utilization provide additional evidence for its relationship to the cellular metabolism of long chain fatty acids.

Staphylococcal pyrogenic exotoxin type C: further characterization.

Staphylococcal pyrogenic exotoxin type C was purified by differential precipitation with ethanol and resolubilization in water followed by thin-layer isoelectric focusing. The purified toxin migrated as a homogeneous protein when re-electrofocused in polyacrylamide (isoelectric point, 7.2), when subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (molecular weight, 22,000 daltons), and when reacted against hyperimmune antisera in an Ouchterlony immunodiffusion plate. A 2200-fold purification of the toxin could be obtained. Pyrogenic exotoxin C was elaborated by all of 44 Staphylococcus aureus isolates from patients with toxic shock syndrome when the strains were tested in a blind manner, but five of 37 control isolates produced toxin (p less than 0.001). Furthermore, all of 91 additional isolates from patients with toxic shock syndrome from diverse geographic locations were positive for toxin. In contrast, 158 of 609 (26%) S. aureus isolates not associated with toxic shock syndrome and one of 22 Staphylococcus epidermidis isolates were positive for pyrogenic exotoxin type C. The biological properties of pyrogenic exotoxin C included pyrogenicity, enhancement of susceptibility to endotoxin shock, enhancement of skin reactivity to give a positive Dick reaction, suppressions of IgM synthesis, and nonspecific T-lymphocyte mitogenicity.

Preparation of an N-acetyl-octapeptide of cholecystokinin : The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N, N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.

Antibodies against viruses and structural brain components in oligoclonal IgG obtained from multiple sclerosis brain.

Immunoglobulin G (IgG) was isolated from three multiple sclerosis (MS) and two control brains by Protein A Sepharose affinity chromatography and was characterized by thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) and antiserum immunofixation. The three MS brains contained oligoclonal IgG. Immunofixation with measles, herpes simplex, varicella, rubella, mumps and cytomegalovirus as antigens and autoradiography revealed that some of the oligoclonal IgG bands separated by PAG IEF contained antibodies against herpes simplex in one, measles in two and varicella virus in all three MS brains. No antibodies were detected with this technique against structural human (crude saline, lipid-proteolipid, ganglioside, and myelin basic protein extracts of MS and normal human brain) and bovine (purified myelin myelin basic protein and oligodendrocytes of bovine brain) brain components. The finding of viral antibodies and the absence of antibodies against structural brain proteins in oligoclonal MS brain IgG is similar to that previously recorded in MS cerebrospinal fluid (CSF).

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.

Elevated immunosuppressive acidic protein in patients with subacute thyroiditis

Immunosuppressive acidic protein (IAP) has been reported to be found in the sera of not only cancer patients but also collagen diseases such as SLE and rheumatoid arthritis and certain inflammatory diseases such as liver abcess and pyothorax. IAP is proved to be one of the heterogeneity of human serum α1 -acid glycoprotein and to suppress both phytohemagglutinin-induced lymphocyte blast formation and mixed-lymphocyte reaction in vitro (Cancer Research, 41 : 3244, 1981).In the present study, we measured IAP by the single radial immunodiffusion methods in patients with various thyroid diseases.The IAP concentration in patients with subacute thyroiditis was 956 ± 256μg/ml (mean ± S.D., n=32), which is significantly higher than that in hyperthyroidism (267 ± 64μg/ml, n=10), hypothyroidism (328 ± 81μg/ml, n=9), chronic thyroiditis (366 ± 120 μg/ml, n=24) and destructive thyroiditis (352 ± 34μg/ml, n=4), respectively. The normal value of IAP concentration is below 500μg/ml.After therapeutic administration of prednisolone or salicylate to patients with subacute thyroiditis, IAP rapidly decreased to normal levels in association with the recovery of elevated erythrocyte sedimentation rate (ESR) and serum thyroxine (T4) and triiodothyronine (T3).In the densitometric tracing of IAP separated by thin-layer isoelectric focusing at a pH range of 2.5 to 5, the major acidic protein had an isoelectric point (pI) of 3.0 in patients with subacute thyroiditis during the active phase as well as in one patient with gastric cancer. Furthermore, the acidic protein at pI 3.0 decreased and the one at pI 3.1, which is the major protein observed in normal serum, became prominent during the recovery phase in patients with subacute thyroiditis. The major acidic protein also had a pI of 3.1 in the sera of patients with hyperthyroidism, hypothyroidism and chronic thyroiditis.IAP remained within the normal range in a patient with destructive thyroiditis who showed thyrotoxic symptoms and then became hypothyroid.It is suggested that the determination of IAP is useful in the clinical diagnosis of subacute thyroiditis. Further analysis of IAP might be helpful for the clarification of the pathogenesis of subacute thyroiditis.