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At least 19 recordsLinked to original sources
PubMed1992

Determination of transcription factor isoelectric point by two-dimensional native isoelectric focusing and electrophoretic mobility shift analysis.

A rapid, sensitive, and inexpensive assay is described for the determination of isoelectric points of native transcription factors derived from cell nuclei. This assay depends on a transcription factors' ability to bind DNA with high specificity and obviates the need for specific antisera or additional detection methods in identifying a particular protein following isoelectric focusing. This method has been applied to two ubiquitous proteins, the octamer transcription factor, Oct-1, and the multisubunit CCAAT box factor, NF-Y. Isoelectric points have been determined under native conditions using conventional isoelectric focusing in the first dimension, followed by binding to a specific oligonucleotide DNA probe, and separation of specific DNA/protein complexes from unbound DNA in the second dimension using native polyacrylamide gel electrophoresis. Oct-1 from HeLa cell nuclei was shown to have a pI of 9.6, while NF-Y was shown to have a pI of 4.5. This method is applicable to any transcription factor which binds DNA specifically and may be used to identify changes in surface charge characteristics which occur as a result of alternative splicing events and/or following transcription factor post-translational modification(s).

Base Sequence↗
PubMed1978

Beta-lactamases: determination of their isoelectric points.

Important discrepancies in isoelectric points (pI) of beta-lactamases were observed depending on the experimental procedure used for their determination: isoelectric focusing (IEF) in sucrose density gradients or analytical IEF in thin-layer polyacrylamide gels (PA). The variations, negligable in the case of TEM-like beta-lactamases, appeared to be important in the case of cephalosporinases and are related to an artifact which appears in PA-IEF. This has been clearly shown with beta-lactamase preparations from the following bacterial strains: Pseudomonas aeruginosa NCTC 8203 (pI = 8.7), P. aeruginosa NCTC 10701 (pI = 9.4), and Proteus morganii NCTC 235 (pI = 8.3). The data previously obtained by PA-IEF were much lower.

Amidohydrolases↗
PubMed1979

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.

Cell Fractionation↗
PubMed1975

Relationship between in vivo degradative rates and isoelectric points of proteins.

Previous studies have shown that in mammalian cells proteins of large molecular weight are degraded more rapidly than small ones. Evidence is presented here that half-lives of proteins are also related to their isoelectric points. A double-isotope method was used to compare degradative rates of soluble proteins separated by isoelectric focusing. In rat liver, skeletal muscle, kidney, and brain, more rapid rates of catabolism were found for acidic protein fractions than for neutral or basic ones. Acidic proteins also tended to be degraded faster in several mouse tissues. A literature survey confirmed this trend. For 22 proteins from rat liver, a highly significant correlation was found between rates of degradation and isoelectric points (r = 0.824; P less than 0.01). This relationship between isoelectric point and half-life appears to be distinct from that between protein size and half-life.

Animals↗
PubMed1979

Heterogeneity of delta-crystallins of the embryonic mallard lens. Correlation between subunit compositions and isoelectric points.

delta-Crystallins from the lenses of embryonic mallards (Anas platyrhynchos) were analyzed with respect to native and subunit molecular weight, subunit composition, and isoelectric point. NaDodSO4-urea-polyacrylamide gel electrophoresis showed that unfractionated mallard delta-crystallins are composed of approximately equal amounts of subunits with molecular weights near 47 000 and 48 000. Agarose gel chromatography showed that the embryonic mallard delta-crystallins have native molecular weights slightly less than 200 000. Thus, embryonic mallard delta-crystallins appear to be tetramers. Five major and nine minor delta-crystallins were resolved by isoelectric focusing. The five predominant delta-crystallins each cross-reacted with antichick delta-crystallin antiserum, and each had a different proportion of the larger and smaller subunits, indicating a direct relationship between the isoelectric point and the subunit composition. The presence of numerous, minor species of native delta-crystallins with different isoelectric points suggested that the subunits possess charge heteogeneity as well as size heterogeneity.

Animals↗
PubMed1979

A releationship between protein-degradation rates in vivo, isoelectric points, and molecular weights obtained by using density labelling.

1. Half-lives of five plant enzymes were estimated by rate-labeling with 2H2O on the assumption that loss of catalytic activity is equivalent to protein degradation. 2. This involved measuring band-broadening of activity profiles after isopycnic centrifugation. 3. Isoelectric points were determined by isoelectric focusing, and molecular weights were estimated by gel filtration. 4. The conclusion is drawn from the experimental evidence presented that a weak correlation exists between rates of degradation and isoelectric points (r = 0.699; P greater than 0.10; not significant). 5. A highly significant relationship exists between the logarithm of subunit size and half-life (r = -0.939; P greater than 0.02). 6. A literature survey confirmed the trends observed.

Centrifugation, Isopycnic↗
PubMed1979

The isoelectric point of thrombin and its behaviour compared to prothrombin at some solid surfaces.

We have shown that the isoelectric point of thrombin is high and that thrombin is a cation at the pH of blood. On the other hand, prothrombin has a low isoelectric point, being more anionic at the pH of blood. It was also found that thrombin adsorbs readily to surfaces, especially negatively charged surfaces, like behenic acid surfaces at pH 8.2. Furthermore, thrombin adsorbed onto behenic acid was active in the sense that it coagulated fibrinogen. The significance of the electric charge of the thrombin molecule in the mechanism of atherosclerosis is discussed.

Adsorption↗
PubMed1978

Large scale isoelectric focusing analysis of the non-histone proteins of the nucleus: isolation of components with alkaline isoelectric points.

After large scale isoelectric focusing of rat liver non-histone protein in polyacrylamide gel, pH range 4--8.6, the only protein material found outside the gradient was present in the cathode solution (20 mM NaOH). This was low mol. wt protein material (approximately 10,000) with an acidic amino acid composition. It bound 5--6 times its own weight of basic ampholine carrier ampholytes to give a complex with a pI of 8.82. This could be dissociated by dialysis against 1 M NaCl.

Animals↗
PubMed1979

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points.

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.

Animals↗
PubMed1992

Isoelectric point of an ion-penetrable membrane.

The surface potential of an ion-penetrable planar membrane is calculated for the case in which acidic and basic groups are present in the membrane. It is found that when both acidic and basic groups are not uniformly distributed in the direction normal to the membrane, the isoelectric point (the pH value at which the surface potential becomes zero) of the membrane varies with the electrolyte concentration, whereas if both groups are uniformly distributed, the isoelectric point is independent of the electrolyte concentration. As a simple example, we treat a membrane consisting of two layers, in which acidic groups are distributed in the outer layer and basic groups are in the inner layer. Simple equations determining the membrane surface potential as a function of pH and electrolyte concentration and the dependence of the isoelectric point on the electrolyte concentration are presented.

Chemical Phenomena↗
PubMed1976

[On the epidemiology of Dientamoeba fragilis Jepps and Dobell 1918. 4th communication: evidence of Dientamoeba fragilis in Enterobius eggs using isoelectric point determination].

By means of the method of determining the isoelectric point, the same conditions of absorption and stain intensity as in Dientamoeba cultivation forms were determined in cells found in Enterobius vermicularis eggs of Dientamoeba fragilis carried. By that way, identity of both stages, as well as function of pinworms as Dientamoeba fragilis vectors were confirmed.

Animals↗
PubMed1975

Role of IgG fractions with high isoelectric points in the thymol turbidity test in syphilis. Evidence for an increase in basic IgG in early syphilis.

It is demonstrated that IgG fractions with isoelectric points above 7.6, isolated from pooled syphilitic sera, are able to elevate the thymol turbidity. The effect increases with increasing isoelectric points of the isolated fraction. IgG from individual syphilitic patients exerts a stronger effect on the thymol turbidity than normal IgG. It is concluded that elevated amounts of these basic immunoglobulins are present in the sera of syphilitic subjects.

Electrophoresis↗
PubMed1978

The isoelectric point of the p30 polypeptide as a marker of mouse endogenous viruses.

The isoelectric point (PI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.

Animals↗
PubMed1992

Molecular connectivity model for determination of isoelectric point of amino acids.

A molecular connectivity model for the calculation of the isoelectric point of amino acids is presented. The model requires calculation of the molecular connectivity values of only the functional fragments of the amino acids. Multiple linear regression with two variables shows the best fit between experimental and calculated data.

Amino Acids↗