Alkaline Isoelectric Point Research Articles

Purification and partial characterization of a high molecular weight metalloproteinase from the venom of Crotalus adamanteus (eastern diamondback rattlesnake)

Chromatography of Crotalus adamanteus venom on CM-Sepharose, Cibacron Blue-Sepharose and Phenyl-Sepharose, followed by gel filtration on Ultrogel AcA 44, has resulted in the isolation in homogeneous condition of a metalloproteinase active on casein and hide powder azure. The proteinase has an alkaline isoelectric point, and the trivial name proteinase B (‘basic proteinase’) is suggested to distinguish it from previously characterized C. admanteus metalloproteinases. Proteinase B is a single chain glycoprotein containing one free sulfhydryl group and having a molecular weight of 60,000. Proteinase B was inactivated by treatment with EDTA, but exposure to phenylmethylsulfonyl fluoride had no effect on proteolytic activity. Proteinase B lacked hemorrhagic activity and did not digest chromogenic substrates specific for thrombin, plasmin or plasma kallikrein.

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa.

Different profiles of isoelectric avian luteinizing hormone components in biological activity and immunoreactivity.

Chicken LH components from the glycoprotein fraction of the anterior pituitary glands have been separated to isoelectric homogeneity by means of isoelectric focusing, and investigated for their biological activities in vitro. The activities of these components were measured with LH receptor binding, cyclic AMP accumulation and testosterone production in rat Leydig cells at equal doses expressed as immunoreactivity of IRC-2 (Gunma). All the components were active in the heterologous assay systems, although the relative potency expressed as the ratios of biological activity to immunoreactivity (B:I) differed significantly among the components. Component I, the amount of which is the largest (40-50%), with the most alkaline isoelectric point (pI) showed the highest B:I ratio, and the B:I ratio decreased with decreasing pI in the same way as in rat LH components (Wakabayashi, 1980; Hattori et al., 1983). Therefore, pituitary LH from photostimulated male quail, where the relative amount of component I was increased, was estimated to have higher B:I ratios than that from short-day (SD) treated male quail. Furthermore, the differences in activities among the components obtained by the three assays were in the following order: testosterone production greater than cyclic AMP accumulation greater than receptor binding, suggesting that the hormonal actions of components with higher B:I ratios (I, II and III) are efficiently amplified in the biological response to the final step. In the incubation of pituitary glands with hypothalamic extracts, component I in the pituitary glands from the long-day (LD) treated group was mostly decreased after the incubation, while all the components were decreased in parallel in the SD-treated group. The results suggest that the LH component releasable to GnRH changes in endocrine status though component I with the highest B:I ratio is relatively releasable in both SD- and LD-treated groups.

Preparative isotachophoresis in a flat bed of granulated gel. I. Principles and procedures, comparison with isoelectric focusing and application to the isolation of a low pI/high mobility form of cat liver cytosolic glutathione S‐transferase

AbstractA method is reported for the separation and partial purification of proteins on a preparative scale (around 100 mg or more total protein per separation run), using the isotachophoretic principle. The technique was carried out using a granulated gel (Ultrodex) as supporting medium, with conventional flat‐bed electrophoresis equipment. This method was applied to the separation of multiple forms of glutathione S‐transferase, derived from the soluble cytosolic fraction of cat liver homogenate. Of particular interest in the present work was the isolation of the highest mobility form of the enzyme, which also functions as a cytosolic ligand‐binding protein for bilirubin. The advantages and disadvantages of isotachophoresis are compred with those of isoelectric focusing, using a simliar apparative set‐up. The latter had the primary drawback that some forms of the enzyme, particularly those with most acidic or alkaline isoelectric points, were, to a certain extent, irreversibly inactivated following focusing, whereas isotachophoresis yielded considerably higher volume activities and resulted in less inactivation during separation. The loss in activity of some forms of the enzyme was particularly marked in isoelectric focusing in thinner gels with agarose as supporting phase. Isotachophoresis has become associated with quite complicated instrumental requirements. The simple form of this technique reported in the present work should provide a useful addition to the spectrum of methods available for protein separation and purification. The major limitation of the flat‐bed configuration is that the actual resolution obtained cannot be fully exploited owing to the technical difficulties associated with sectioning the flat‐gel.

Sialic Acid Dependent Polypeptide Chain Heterogeneity of Human Fibrinogen Demonstrated by Two-Dimensional Electrophoresis

Human fibrinogen was compared with asialofibrinogen by two-dimensional electrophoresis to evaluate the contribution of sialic acid to the heterogeneity of the gamma- and B beta-polypeptide chains. Reduced fibrinogen showed three major variants for both the gamma- and B beta-chains. In addition two minor gamma-bands with a more acidic isoelectric point than the normal gamma-chains were observed. Electrophoresis in the second dimension (SDS) suggests that these most acidic bands are gamma-chain-variants with a higher molecular weight. In asialofibrinogen only two predominant variants with more alkaline isoelectric points were present in each chain type. It is concluded that enzymatic removal of sialic acid partially reduced the heterogeneity of the gamma- and B beta-polypeptide chains of human fibrinogen, but additional sources producing charge heterogeneity must be sought.

Anamalous electrophoresis behavior of rebonuclease from human urine and a novel treatment method for reliable analysis

AbstractSince Desialylated rebonuclease (RNase) from human urine has a highle alkaline isoelectric point (pI 9.5–10.5), an acidic buffer system is required for electrophoretic analysis of different urine specimens. In a common acid disc electrophoresis system in thin‐layer polyacrylamide gel, the cathodal mobility of the RNase enzyme(s) was markedly reduced from that expected on the basis of the estimated pI. Dialysis urine against water had a very detrimental effect on the separation of the isozymes, and the addition of buffer to the dialyzed urine further exacerbated this effect. The urin RNase exhibited anomalous electrophoretic behavior in the form different urine specimens or from the same urine specimen adjusted to different concentrations. Treatment of urine with various cations had a significant, positive effect on the migration and resolution of the isozymes. This and other experimental evidence is consistent with the hypothesis that the anomalous behavior was due to the binding of anions from the buffer. A standardized protocol was developed for processing urine specimens, including treatment of urine with myoglobin, which abolished the artifactual variation observed previously and enabled reliable electrophoretic separation of the urine RNase isozymes. In addion, myoglobin treatment had a similar positive effect on the electrophoretic behavior of RNase enzymes from some other sources.

A bifunctional enzyme from glyoxysomes. Purification of a protein possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities.

1. Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase copurified when extracts from cotyledons of 5-day-old cucumber seedlings were fractionated by acetone precipitation, ion-exchange chromatography on CM-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. 2. Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM-cellulose, or blue-dextran-Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein. 3. Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined. 4. The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of beta-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.

Origin of multiple species of yeast enolase A on isoelectric focusing

On isoelectric focusing, bakers yeast enolase A has been shown to resolve into one major and at least two minor species. Refocusing of individual species isolated by electrofocusing shows the minor species to be formed from the major one. The extent of formation of the minor species increases with electrofocusing time and decreasing quantity of protein electrofocused, suggesting that the protein, on dissociating, tends to change into forms(s) with more alkaline isoelectric points.

Apparent antihaemophilic activity of basic amphoteric polyelectrolytes.

Amphoteric polyelectrolytes with alkaline isoelectric points (for example Ampholine 9-11) intended for use in isoelectric focussing were found to shorten the prolonged partial thrombosplastin clotting time with kaolin (PTTK) of plasma deficient in factor VIII. The response of this PTTK to Ampholine 9-11 was linear when plotted on double log paper with a slope slightly greater than that of factor VIII itself. Ampholine 9-11 at 2% concentration had factor VIII and factor IX activity equal to that of normal plasma as well as somewhat less activity as factor XI. It had no significant correcting effect on the clotting of plasmas deficient in factors V, VII, X, II OR XII. Ampholine 9-11 inhibited low concentrations of contact activators and its effect in correcting factor VIII deficient plasma was found to be related to the degree of contact activation.

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.

Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons

Glyoxysomes were isolated from etiolated cotyledons of cucumber seedlings. After separation of matrix proteins from the glyoxysomal membranes, enzymes were solubilized from the membranes by 100 m m MgCl 2 and purified by sedimentation velocity centrifugation, ion exchange chromatography, and separation on hydroxylapatite. Malate synthase, citrate synthase, and malate dehydrogenase the three enzymes of the glyoxylate cycle which were primarily membrane bound in this type of microbody-were thus obtained in a homogeneous form, as judged by sodium dodecyl sulfate-gel electrophoresis. Enzymatically active malate synthase, as obtained by solubilization of membrane proteins, behaved on Sepharose 6B columns as a protein with a molecular weight of about 70,000 and is characterized by an acidic isoelectric point. Malate synthase aggregates in the presence of Mg 2+ and glyoxylate, yielding an active octamer with an alkaline isoelectric point and a molecular weight of about 540,000. Upon sodium dodecyl sulfate-gel electrophoresis, a subunit molecular weight of 63,000 was estimated. Citrate synthase exists as a dimer (molecular weight of 100,000) and tetramer (molecular weight of 200,000) and exhibits the same subunit molecular weight as the liver enzyme (46,000). Malate dehydrogenase was found to have a molecular weight similar to the microbody catalase (about 225,000), while for the single peptide chain a value of approximately 34,000 was determined.

Separation and comparison of isoenzymes of [formula omitted] of pregnancy serum by polyacrylamide gel electrofocusing

Sera from normal and pregnant subjects as well as those from subjects with Tay-Sachs disease, hyperlipidemia and cancer were subjected to electrofocusing in the pH range of 3–10 in polyacrylamide gel (7.5%). The separated isoenzyme bands were stained for visualization on the gel for activity with α- naphthol-AS-BI-N- acetyl-β- D-glucosaminide as substrate. Elevation of N- acetyl-β- D-glucosaminidase (hexosaminidase) activity during pregnancy was found to be due to a progressive increase in number of isoenzymes: 1–2 bands in the early stage of pregnancy (5 weeks) to 6–8 bands in the last trimester. By comparison with the known isoenzymes of human aortic hexosaminidase, at least one of these bands corresponds to the A form, 4–5 bands to the P form and 1–2 bands to intermediate between the A and P forms. In the B form region only a single band with weak enzyme activity was observed, and its intensity of staining remained almost unchanged during pregnancy. Heat treatment (50° for 3 h at pH 4.4) resulted in inactivation of isoenzymes in the area on the gel of the A form and of the intermediates between the A and P forms but had no effect on the P and B forms. However, some bands found in the A area, between the A and P areas and in the P regions, transformed into forms having more alkaline isoelectric points (pIs) by this treatment. The new forms were found predominantly in the B area. The proportion of the thermostable (P and B) and the thermolabile (A) forms of the enzyme by such treatment was 32% and 68%, respectively, for normal serum, and 70% and 30%, respectively, for pregnancy serum. The value for pregnancy serum remained fairly constant throughout pregnancy (5 weeks to the last trimester). Tay-Sachs serum (one case) possessed three very minor bands corresponding to the P region and two major bands in the B region. When heat-treated, a partial transformation of minor bands and a complete transformation of one of the major bands in the B region occurred. One B band was transformed into the other B band having the most alkaline pI. Total hexosaminidase activity, meanwhile, did not show any change. Increase in number of isoenzymes of hexosaminidase in the P region was also found in sera of hyperlipidemic and cancer patients, suggesting that the P form may not be specific to pregnancy.