Isoelectric Focusing Slab Gel Research Articles

13. The β-hexosaminidase story in Toronto: From enzyme structure to gene mutation

This chapter discusses the structure of Hexosaminidase A and Hexosaminidase B. It was found that while the β subunits of Hex A and Hex B contained two polypeptides of 25–30 kDa, the β subunit of Hex A was composed of a single chain of 50–60 kDa.A and Hex B did contain two polypeptides of 25–30 kDa, the β subunit of Hex A was composed of a single chain of 50–60 kDa. The reduced and alkylated Hex A and Hex B are separated on isoelectric focusing slab gels (IEF-PAGE), run in urea and detergent, and the polypeptide chains forming the β subunit in both isozymes could be separated in an acidic group and a basic group of heterogeneous bands. The chapter also discusses isolation of cDNA clones coding for α and β chains, homology between the deduced α and β primary structures, posttranslational processing of the pre-pro-α and pre-pro-β chains, structure–function relationships, and molecular heterogeneity in Tay-Sachs and Sandhoff Diseases.

One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels

This unit describes isoelectric focusing (IEF) of polypeptides in urea-containing polyacrylamide slab gels as a result of a pH gradient created by ampholytes (small charged organic molecules) in the gel in response to an electric field. Separation on the basis of isoelectric point produces bands of polypeptides that can be electrotransferred to suitable membranes for further analysis, for example, by staining or immunodetection. If samples have been radiolabeled, they can be visualized by fluorography. This unit contains support protocols detailing special conditions for application of denaturing IEF slab gels for electrotransfer and fluorography in which a scintillator is incorporated into the gel after completion of the run to facilitate detection of radioactively labeled polypeptides.

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to, the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.

Two‐dimensional electrophoresis of membrane proteins: Separation of myelin proteins

A method for two-dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X-100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab gel before separation. Subsequent discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting, the positions of myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase were established with specific antibodies.

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.

Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II.

Two previously unidentified apolipoproteins (apo) designated apo C-II-X and C-II-Y have been found in plasma of homozygotes and obligate heterozygotes of a family with apo C-II deficiency. Because the plasma of homozygotes do not activate lipoprotein lipase, apo C-II-X and C-II-Y are apparently nonfunctional. These apolipoproteins have isoelectric focusing points of 5.15 and 5.54, respectively, compared with 4.88 and 4.74 for the known isomorphs, C-II-1 and C-II-2, respectively. They have approximately similar molecular weights to apo C-II-1 and C-II-2 on two-dimensional sodium dodecyl sulphate-glycerol-polyacrylamide slab gel electrophoresis. They do not form insoluble antigen-antibody complexes with antibodies to apo C-II in single antibody immunodiffusion or electroimmunoassay systems. However, using a double-antibody technique in which immunoblotting is coupled with polyacrylamide isoelectric focusing slab gel electrophoresis, apo C-II-1, C-II-2, C-II-X, and C-II-Y have similar reactivity with antibodies to apo C-II. In this family the presence of apo C-II-X and C-II-Y discriminates obligate heterozygotes from normal subjects.

Comparison of liver alcohol dehydrogenases in Fischer-344 and Sprague-Dawley rats

1. 1. Livers of Sprague-Dawley rats contain 30–100% more alcohol dehydrogenase activity than livers of Fischer-344 rats. 2. 2. When weight-matched rats from both strains are injected with the same dose of ethanol (1.2 g/kg), Sprague-Dawley rats achieve lower blood alcohol levels than Fischer-344 rats at all the time-points tested. 3. 3. Purified alcohol dehydrogenases from both strains of rats exhibit identical electrophoretic mobilities in SDS-polyacrylamide§ and in isoelectric focusing slab gels, pH optima, peptide maps, K m for ethanol, and capacities to bind monospecific rabbit antibodies. 4. 4. Quantitative differences in alcohol dehydrogenase activity between these strains of rats are due to differences in their liver alcohol dehydrogenase levels.