Isoelectric Focusing In Granulated Gel Research Articles

CLASSIFICATION OF GREEN PEA PEROXIDASES BY PREPARATIVE ISOELECTRIC FOCUSING

Green pea peroxidase isoenzymes were partially purified, identified, and grouped by preparative isoelectric focusing in granulated gel (PEGG), using a modified cascade method. Isoenzyme patterns were detected utilizing a substrate-impregnated paper print technique. Direct application of crude extract on PEGG proved to be a valuable tool for partial purification of peroxidase isoenzymes. A substrate-impregnated paper print technique enabled the detection of isoenzyme patterns and may replace the time-consuming conventional peroxidase activity assay. Twelve isoenzymes (pIs ranging from 5.5–8.6), isolated on analytical thin-layer isoelectric focusing on polyacrylamide gel, were isoelectrically homogeneous.

Characterization of cationic albumin in minimal change nephropathy

The presence of isoalbumins with a less anionic charge than the normal protein (pI = 4.7) is the hallmark of proteinuria in minimal change nephropathy (MCN). Steroid-induced restoration of near normal levels of proteinuria is characterized by the appearance in urines of isoalbumins with a pI still more anionic than the normal. In our search for an explanation for the pI changes, we used preparative isoelectric focusing in granulated gels to split the microheterogeneous bands obtained from nine MCN-affected children into four fractions (A1, A2, A3, A4) with decreasing pI from 5.8 to 4.0 and we have determined their fatty acid content. The least anionic fraction, A1, was the most defatted, followed by A2, A3 and A4 in which fatty acid content progressively increased, A4 being the most fatted fraction. Accordingly, the mean content of fatty acids in urinary albumin in proteinuric children was lower than in both the remission phase and in normal children (2.17 +/- 0.03 vs. 20.91 +/- 0.38 and 20.94 +/- 0.39, respectively) and was lower by a factor of 4 compared to serum albumin in the same phase of the disease (2.17 +/- 0.03 vs. 8.59 +/- 1.64). Among medium and long-chain fatty acids, the ratio between serum and urinary albumin was the highest for linoleic acid (approximately 7), followed by that of oleic acid, palmitic acid and lauric acid. At variance in five other patients affected by non-MCN nephrotic syndrome this ratio was for practically all FAs about 1.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo studies of iron delivery by human monoferric transferrins.

According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. In this study we present the results of an evaluation of this hypothesis in vitro and in vivo with human pure monoferric transferrins obtained by preparative isoelectric focusing in granulated gels. The uptake of iron from monoferric transferrins TfFeC and FeNTf by erythroid bone marrow cells, hepatocytes and stimulated T-lymphocytes in vitro was equal, even when both monoferric transferrins were present together in the incubation medium. Ferrokinetic studies in vivo, performed with both pure monoferric transferrins, showed that transferrin TfFeC, as well as transferrin FeNTf, mainly deliver their iron to the erythron. As red cell 59Fe utilization, red cell iron turnover and other ferrokinetic parameters, obtained from this study, were identical too it is evident that both iron-binding sites of transferrin are functionally homogeneous in vivo, with respect to iron delivery.

Purification and characterization of lectins from Vicia hirsuta

The presence of three lectins in the seeds of Vicia hirsuta (L.) S. F. Gray, a wild‐growing vetch, was shown. The main lectin was purified to homogeneity by buffer extraction, ammonium sulfate precipitation, affinity chromatography on Sephadex G‐100 and isoelectric focusing in granulated gel. By chromatofocusing instead of isoelectric focusing the yield was increased 5‐fold. The lectin has a pi of 6.4. It is composed of large β‐subunits (Mr 19.200) and small α‐subunits (Mr 12.800) in a 1:1 ratio. The subunits can be separated on Sephadex G‐75 when equilibrated with 6 M guanidine‐HCl. The amino acid composition of the two different subunits has been determined. No sulfur‐containing amino acids are present. The lectin resembles the lectins from legumes from the same cross‐inoculation group, i.e. Lens culinaris, Lens esculenta, Pisum satiyum and several Vicia spp. by the same type of sugar specificity and amino acid composition.

Evidence of the Existence of a Factor That Induces C3 Receptors on Bone Marrow Cells

Abstract The existence of a molecule responsible for the induction of C3R receptors (C3R) on murine bone marrow cells (C3R inducer, C3RI) is demonstrated in lung conditioned media. The inducer of Fc receptors on murine bone marrow cells (FcRI) and the colony-forming factor MGI (macrophage and granulocyte inducer) are also identified from the same source. The three inducing factors are shown by molecular sieving chromatography and by isoelectric focusing in granulated gel to be three different molecules. The molecular weights estimated were of 35,000, 22,000, and 10,500 for C3RI, MGI, and FcRI, respectively, with isoelectric pH values of 3.9, 6.6, and 7.6 for the same molecules. The C3RI is assumed to be of protein nature, because the molecule lost its biologic activity after trypsin or heat treatment. In the two techniques used, the three peaks of activity were completely separated, suggesting that the induction of C3 or Fc receptors and colony formation are mediated by different molecules and thus regulated independently at the cellular level.

Evidence of the existence of a factor that induces C3 receptors on bone marrow cells

The existence of a molecule responsible for the induction of C3R receptors (C3R) on murine bone marrow cells (C3R inducer, C3RI) is demonstrated in lung conditioned media. The inducer of Fc receptors on murine bone marrow cells (FcRI) and the colony-forming factor MGI (macrophage and granulocyte inducer) are also identified from the same source. The three inducing factors are shown by molecular sieving chromatography and by isoelectric focusing in granulated gel to be three different molecules. The molecular weights estimated were of 35,000, 22,000, and 10,500 for C3RI, MGI, and FcRI, respectively, with isoelectric pH values of 3.9, 6.6, and 7.6 for the same molecules. The C3RI is assumed to be of protein nature, because the molecule lost its biologic activity after trypsin or heat treatment. In the two techniques used, the three peaks of activity were completely separated, suggesting that the induction of C3 or Fc receptors and colony formation are mediated by different molecules and thus regulated independently at the cellular level.

A rapid procedure for the isolation of human sex steroid binding protein

A three stage procedure is described which provides a rapid, simple, and reliable means to isolate sex steroid binding protein from human serum. Purification is achieved by the sequential use of affinity chromatography, isoelectric focusing in granulated gels and ion exchange chromatography. Between 3–4 mg of protein can be recovered from one litre of pregnancy serum without four days. A molecular radius of 2.98 nm and an apparent molecular mass of 91000 were obtained for the purified native protein by polyacrylamide gel electrophoresis. A value of 50000 was obtained for the molecular mass of the reduced protein by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The purified protein was used to prepare a monospecific rabbit antiserum and a murine monoclonal antibody. Partial characterisation of the latter has been achieved.

No functional difference of the two iron-binding sites of human transferrin in vitro.

1. According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. 2. The aim of the study presented was to evaluate this hypothesis in a homogeneous system, with human bone marrow cells and pure human monoferric transferrins A and B. 3. For this reason normal human bone marrow cells were incubated with human monoferric transferrin. The monoferric transferrins A and B were obtained by selective labelling at different pH of apotransferrin followed by preparative isoelectric focusing in granulated gels. The uptake of iron by the cell suspensions from monoferric transferrins A and B was equal. 4. In a heterogeneous but more active system for the removal of iron from human transferrin in vitro the two human monoferric transferrins did not show any significant functional differences. 5. No support for the Fletcher-Huehns hypothesis could be obtained.

Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E.

Moloney leukemia virus activated both the classical and alternative pathways of human complement. About 500,000 virions were required to detect activation of the classical pathway whereas 5,000 times as many virions were necessary to initiate the alternative pathway, indicating that in this system only the former is of biological significance. Disruption of the virus with Triton X-100 destroyed its ability to initiate the alternative pathway without affecting its ability to activate the classical pathway. After ultracentrifugation of disrupted virus the active component could be recovered in the supernate and was isolated by isoelectric focusing in granulated gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic and analysis and cyanogen bromide digestion studies revealed that the activity resided in a methionine-containing protein having a pI of 7.5 and a molecular weight of approximately equal to 15,000 daltons. The purified protein interacts strongly with Clq and efficiently activates Cl. RNase and lipolytic enzymes had no effect on the isolated protein but incubation with trypsin resulted in loss of activity. Enzymatic digestion studies of surface-labeled virus indicate that the active protein is a viral membrane protein. On the basis of these results it is concluded that the complement receptor of Moloney leukemia virus is the surface protein p15E.