Isoelectric Focusing Properties Research Articles

Low-molecular-mass colored compounds for fine tracing of pH gradient on broad and narrow scale in isoelectric focusing

We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds’ pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.

Isolation of a crotoxin-like protein from the venom of a south american rattlesnake ( Crotalus durissus collilineatus)

1. 1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. 3. Results indicate that “collilineatus toxin” is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.

Comparison of labeled heat shock proteins in neuronal and non-neuronal cells of Aplysia californica

Aplysia californica has been used to study the protein synthetic response of nervous tissue to stress induced by elevated temperatures. The abdominal and pleural ganglia as well as associated connectives were exposed to various temperatures for 30 min, labeled with [33S]methionine at room temperature, and then analyzed by sodium dodecyl sulfate gel electrophoresis. All cells examined responded to temperatures of greater than 31 degrees C by a reduction in levels of labeled actin, as well as by the enhanced labeling of proteins with apparent Mr of 70,000 and 110,000. Two-dimensional electrophoresis indicated that the molecular weight and isoelectric focusing properties are similar to the heat shock proteins (HSPs) observed in other systems. In addition to these major HSPs, heat-induced proteins with molecular weights ranging from 70,000 to 90,000 were highly labeled in the neurosecretory bag cells. Further cell type-specific differences in the protein synthetic response to elevated temperatures were revealed by quantitation of the major HSPs. Levels of labeled HSPs were significantly lower in ganglion cells as compared to the non-neuronal connective cells. In addition, the decrease in actin levels appeared to be less dramatic in the ganglion cells. Analysis of the cellular compartmentalization of HSPs suggests that both neurons and glia are capable of HSP synthesis. Studies in the squid have demonstrated that HSPs are transferred from adaxonal glia into the axoplasm (Tytell, M., S. G. Greenberg, and R. J. Lasek, unpublished observation).(ABSTRACT TRUNCATED AT 250 WORDS)

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as though they were ~ M r 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 m guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.

Solubilization, stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K m values (0.2–0.4 mM) for the synthetic fluorometric substrate 4- methylbelliferyl-α- D -N- acetylneuraminic acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (p I values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.

Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of the major phosphorylation sites for cAMP-dependent protein kinases.

Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes.

Characterization of α2-macroglobulin from plasma of cystic fibrosis patients and controls

The physical, kinetic, and isoelectric focusing properties of native α 2-macroglobulin from cystic fibrosis and control plasmas were studied. No differences were found in the esterolytic activity levels of control, obligate heterozygote, and cystic fibrosis plasmas. Stability studies indicated that both control and cystic fibrosis α 2-macroglobulin retained full activity for at least 8 months at −20°C, a week at 0–4°C, 11 hr at 50°C, and showed no differences in thermostability at several preincubation temperatures. The microheterogeneity of native α 2-macroglobulin was studied by column isoelectric focusing of five control and five cystic fibrosis plasmas. The number and p I values of the isoelectric forms between pH 4.5–8.0 were quite similar for both groups even though consistently less cystic fibrosis α 2-macroglobulin activity was recovered after isoelectric focusing.

Differential isoelectric focusing properties of crude and purified human α 2-macroglobulin and α 2-macroglobulin—proteinase complexes

The isoelectric focusing (IEF) properties of human α 2-macroglobulin (α 2M) and α 2M—proteinase complexes from crude and partially purified preparations were studied by column IEF. The average isoclectric point (pI) of the major form was 6.5 for α 2M from crude plasma but was 5.3 for purified samples. Following preincubation with either trypsin, chymotrypsin or pancreatic elastase the crude α 2M—proteinase complexes displayed p I values ranging from 5.3 to 6.1 and the purified α 2M—proteinase complexes ranged from ph 6.0 to 6.1. A comparison of recoveries for focused crude or purified α 2M and trypsin-preincubated α 2M indicated enhanced recovery for the trypsin-preincubated samples suggesting that the binding of the proteinase enhances the stability of α 2M. α 2M thus displays column IEF properties which appear to be dependent upon the state of purity of the molecule. These findings are of particular significance to investigators concerned with using expressions of altered α 2M electrophoretic patterns for clinical diagnostic purposes in such diseases as multiple sclerosis, diabetes and cystic fibrosis.

Cystic fibrosis serum α- l-fucosidase: Confirmation of normal activity levels and normal kinetic and isoelectric focusing properties

Comparable α- l-fucosidase activity was found for normal and cystic fibrosis sera, 7.8 ± 3.3 and 9.8 ± 4.7 nmol/min/ml, respectively. Isoelectric focusing of normal and cystic fibrosis sera α- l-fucosidase revealed similar isoelectric profiles with most activity between isoelectric points of 4.7–5.4. Kinetic analysis of normal and cystic fibrosis sera α- l-fucosidase revealed identical apparent Michaelis constants for the 4-methylumbelliferyl substrate (51 ± 6 μmol/l and 50 ± 8 μmol/1, respectively), similar pH optima curves (both have broad optima between pH 4.8 and 6.5) and identical thermostability curves at three different preincubation temperatures (37°, 45° and 55°C).

Purification and properties of the .sigma. subunit of Escherichia coli DNA-dependent RNA polymerase

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.

Evidence for specific recognition sites mediating clearance of lysosomal enzymes in vivo.

A study of the clearance of liver lysosomal enzymes was carried out in the rat. Purified rat liver lysosomal beta-D-glucuronidase (EC 3.2.1.31), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alpha-L-fucosidase (EC 3.2.1.51), and alpha-D-mannosidase (EC 3.2.1.24), as well as rat preputial gland beta-glucuronidase, were infused intravenously into anesthetized rats. All of the enzymes were rapidly cleared from the circulation. Sodium periodate oxidation of lysosomal beta-glucuronidase resulted in a near abolition of rapid clearance, a reduction in concanavilin-A-Sepharose binding, and a reduction in neutral sugar content, accompanied by alteration in isoelectric focusing properties. Similarly, periodate oxidation of lysosomal N-acetyl-beta-D-glucosaminidase resulted in a loss of the rapid clearance property. These results suggest that specific recognition sites occur on lysosomal hydrolases which mediate clearance following intravenous injection, and that these sites involve the carbohydrate portions of the enzymes.

Genetics of restricted antibodies to streptococcal group polysaccharides in mice. I. Strain differences of isoelectric focusing spectra of group A hyperimmune antisera.

The immune response of nine inbred and one outbred strain of mice to the streptococcal group A polysaccharide was investigated with respect to magnitude and restriction. Analytical isoelectric focusing served as a tool to estimate the degree of restriction of Group A polysaccharide-specific antibodies. It proved feasible to distinguish low and intermediate from high responder strains, and to delineate strain-specificity of isoelectric focusing spectra of the immune sera. For example, immune sera of BALB/c mice, restricted high responders, and of C57BL/6 mice, heterogeneous low responders, had distinct focusing properties. Responsiveness was a dominant autosomal genetic trait in C57BL/6 x BALB/c F(1) hybrid mice, irrespective of the maternal and the paternal genotype; the immune sera of these mice had their own, rather uniform isoelectric focusing spectra whereby structural genes of the low responder strain were expressed to predominant levels in 81% of the hybrids. Responsiveness in C57BL/6 x BALB/c F(2) progeny segregated into 79% high and 21% low responders, and showed no genetic linkage to the following characteristics: hair color, sex, H-2 type, and Ig allotype of the heavy chain. The isoelectric focusing properties of these immune sera indicated segregation into patterns like BALB/c mice (40%), F(1) hybrids (48%), and C57BL/6 mice (12%). Since this segregation is independent of any of the above criteria in these F(2) mice a regulatory gene(s) is postulated that controls the clonal pattern of the immune response.