Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Identification of calcium binding proteins from brain

A procedure was worked out for purification and identification of calcium-binding proteins from bovine brain using Ca²⁺-dependent, reversible binding to a hydrophobic support, phenyl-Sepharose, as the method of isolation. These proteins could be visualized during and after their separation by running them on non-denaturing polyacrylamide gels, blotting to Zeta-probe paper, and autoradiographing with⁴⁵Ca²⁺. About 24 polypeptides could be seen in this fraction on SDS (Laemmli) gels and about 8–10 native, Ca²⁺-binding proteins could be seen on non-denaturing gels and on blots of their 45Ca²⁺ autoradiographs. Some of these proteins could be purified further by chromatography on DEAE-Sephacel and still retain their⁴⁵Ca²⁺-binding activity.     

Evidence for protein phosphorylation inStreptomyces lincolnensis

Protein phosphorylation was investigated inStreptomyces lincolnensis underin vivo conditions. In cells grown in the presence of³²P-orthophosphate, proteins ofM=12, 22, 45, 68 and 90 kDa were labeled with³²P (detected by gel electrophoresis and autoradiography). These proteins were shown to contain O-phosphoserine and a small proportion of O-phosphotyrosine. Taken together the results indicate thatStreptomyces lincolnensis harbors several protein kinases including a protein-tyrosine kinase activity.     

Regulation of extracellular proteins and α-amylase secretion by temperature inBacillus subtilis

α-Amylase was found to be the main protein secreted byBacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45°C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis.¹⁴C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30°C than that at 40 or 45°C. The effect of temperature on α-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.     

Enhancement of detection for partially methylated alditol acetates by chemical ionization mass spectrometry

Treatment of washed, intact platelets with Bolton-Hunter reagent is a satisfactory method for ¹²⁵I-labeling of many platelet proteins. Analysis by two dimensional polyacrylamide gel electrophoresis and autoradiography shows that the major platelet cytoskeletal proteins and at least four surface-exposed proteins are labeled. The method allows the identification of these labeled proteins in amounts that are below the limits of detection by Coomassie blue staining. Two granule proteins, thrombospondin and fibrinogen, are slightly labeled. Conditions of labeling do not appear to affect platelet structure or function, as assessed by phase-contrast microscopy, ⁵¹CrO₄^2? release, and aggregation in response to thrombin or fibrinogen/adenosine-5′-diphosphate.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Cleavage of cell surface proteins by thrombin

This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin-mediated cleavages using 2-dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: (1) proteins were labeled by lactoperoxidase-catalyzed iodination using ¹²⁵I^?; (2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with ³H-NaBH₄; and (3) glycoproteins were metabolically labeled by incubating cells with ³H-fucose. Labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with ³H-fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin-sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin-sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin-sensitive. However, cell surface proteins of about 45K daltons and 130K to 1 50K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin-mediated cleavages of cell surface proteins in a large number of cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages that are necessary for thrombin-stimulated cell division.     

Characterization of bone protein components with polyacrylamide gel electrophoresis: effects of zinc and hormones in tissue culture

An attempt was made to clarify the molecular characterization of zinc-induced bone protein synthesis in tissue culture. Calvaria were removed from weanling rat (3-week-old male) and cultured for periods up to 48 hr in Dulbecco's Modified Eagle Medium (high Glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. When calvaria cultured in the presence of 10^–5 to 10^–4 M zinc were pulsed with [3H] leucine, zinc caused a significant increase in the incorporation of [³H] leucine into the acid-insoluble residues of bone tissue. The soluble fraction obtained from cultured bone was analyzed with SDS-polyacrylamide gel electrophoresis (SIDS-PAGE). The major components in the fraction obtained from control bone were 68 killo-dalton (kDa) and 45 kDa proteins. These components were clearly increased by the presence of zinc (10^–4 M). The effect of zinc was completely abolished by the coexistence of 10^–6 M cycloheximide. Meanwhile, 10^–9 M estrogen or 10^–8 M insulin, which can stimulate bone formation, did not enhance the effect of zinc to increase bone 68 and 45 kDa proteins. The present findings suggest that zinc increases many bone protein components, especially 68 and 45 kDa proteins.     

Molecular divergence of a major peptidoglycan synthetase with transglycosylase - transpeptidase activities in Escherichia coli — Penicillin-binding protein 1Bs

Penicillin-binding protein 1Bs of Escherichia coli (Mr ca. 9 × 10⁴) gave three protein bands with slightly different mobilities on sodium dodecylsulfate — polyacrylamide gel electrophoresis. The enzymatic activities of each of these proteins were identified after renaturation of the proteins separated by electrophoresis. Each of them had two enzymatic activities of the last steps of synthesis of peptidoglycan from lipid-linked precursor, i. e., activity of transglycosylase, which extends the glycan chain, and activity of penicillin-sensitive transpeptidase, which crosslinks glycan chains with peptide cross-bridges. Trypsin treatment of each of the three proteins resulted in formation of a doublet of penicillin-binding proteins (Mr ca. 5 × 10⁴). The results strongly indicate that penicillin-binding protein 1Bs are bifunctional peptidoglycan synthetase proteins differing slightly in molecular structure.     

Radioenzymatic assay for the acetohydroxy acid synthase-catalyzed synthesis of alpha-aceto-alpha-hydroxybutyrate

A method has been developed for radiolabeling small amounts of ribosomal proteins extracted from polyacrylamide gels with potassium [¹²⁵]Iiodide. The procedure was used to label even those proteins which lack tyrosine and histidine residues by the modification of proteins with methyl p-hydroxybenzimidate. Specific radioactivities obtained range from 20,000 to 200,000 cpm/μg. The method has been used in the identification of eukaryotic ribosomal proteins from rabbit reticulocytes separated by polyacrylamide/sodium dodecyl sulfate gel electrophoresis.     

Diphtheria toxin inhibits the synthesis of myelin proteolipid and basic proteins by peripheral nerve in vitro

Abstract— Diphtheria toxin (DT) did not produce measurable degradation of myelin proteins or sulphatide in sciatic nerves of chick embryos after incubation in vitro for 4 h. In contrast, DT inhibited the in vitro incorporation of L-[U-¹⁴C]leucine into myelin proteins by the nerves after a delay of 1 h. Separation of the myelin proteins by SDS-polyacrylamide gel electrophoresis indicated that the synthesis of Wolfgram proteins and proteins not entering the gel was inhibited by 21–22 per cent, whereas synthesis of myelin proteolipid and basic proteins was inhibited by 79–88 per cent. Incorporation of ³⁵SO₄ into myelin [³⁵S]sulphatide was also inhibited by DT after a delay of 2 h. The inhibition of [³⁵S]sulpha-tide incorporation into myelin caused by DT differed from that observed with puromycin in that it did not depend on depletion of an intracellular transport lipoprotein. Instead, the inhibition seemed to be secondary to the decreased synthesis of myelin proteolipid and basic proteins.     

The stress-70 protein family in diplopods: induction and characterization

To induce stress-70 proteins (hsp70), adults of the millipede Julus scandinavius (Diplopoda) were exposed to leaf litter contaminated with different concentrations of Cd²⁺ (10, 30, 50 and 60 mg·kg^-1 as CdCl₂). The expression of hsp70 was investigated by semiquantitative and qualitative biochemical methods. After SDS-gel electrophoresis and Western blotting a subsequent digital image analysis showed that increasing dietary concentrations of Cd²⁺ resulted in elevated levels of hsp70, which in turn indicated proteotoxic condition. Qualitative results were obtained by two-dimensional gel electrophoresis. A stress-70 protein family, similar to that of other arthropods, was detected in Julus scandinavius: at least five different proteins with an approximate molecular weight of 68, 69, 70, 77, and 78 kDa could be distinguished after heat shock as well as after Cd²⁺ exposure.Abbreviations IEF isoelectric focusing - hsp heat shock protein(s) - grp glucose regulated protein(s) - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Proteome of the bacterium <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis>

Using modern proteomic assays, we have identified the products of gene expression and posttranslational modifications of proteins of the bacterium Mycoplasma gallisepticum S6. Combinations of different technologies of protein separation by electrophoresis and mass-spectrometric analysis gave us a total of 446 proteins, i.e. 61% of the annotated proteins of this microorganism. The Pro-Q Diamond and Pro-Q Emerald dye technology was used for fluorescent detection of ten phosphoproteins and two glycoproteins. The acylation of proteins was studied by electrophoresis after in vivo labeling with different ¹⁴C-labeled fatty acids, followed by autoradiography. Sixteen acylated proteins were identified, with a quarter of them involved in plasma membrane construction and another quarter involved in cell energy metabolism.     

In vivo acylation of Dictyostelium actin with palmitic acid

Cells of Dictyostelium discoideum were incubated with [³H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane-associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti-actin antibody, its coincidence with the major [³⁵S]methionine-labeled protein after two-dimensional electrophoresis and its binding to a DNase I affinity column. The ³H-label was resistant to chloroform-methanol extraction and boiling in SDS-containing buffer. After partial purification by preparative SDS-polyacrylamide gel electrophoresis, the 44-kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas-liquid chromatography.     

High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

Characterization of sulphatide-containing lipoproteins in rat brain

—(1) Water-soluble [³⁵S]sulphatide is found in the 105,000 g supernatant (SN) of rat brain after intraperitoneal injection of Na₂³⁵SO₄. This labelled sulphatide has a density between those of free lipid and free protein. (2) Fractionation of SN by preparative acrylamide gel electrophoresis indicates that the [³⁵S]sulphatide is not distributed among all SN proteins, but is associated with certain specific proteins. One of the isolated [³⁵S]sulphatide-containing proteins appears homogeneous by analytical acrylamide gel electrophoresis at several pH values. (3) Comparison of the turnover of [³⁵S]sulphatide in microsomes, SN, and myelin indicates that these three subcellular fractions behave as distinct metabolic pools, which meet the requirements for a precursor-product relationship between microsomes and SN and between SN and myelin. (4) These results suggest that sulphatide, synthesized in the microsomes, is transported to the myelin membrane as water-soluble sulphatide containing Iipoproteins in SN.     

Regulation of HSP70 and HSP28 gene expression: absence of compensatory interactions

We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [³H] leucine and [³²P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.     

Phosphorylation of Chromatin-associated Proteins in Lemna and Hordeum

Sterile embryos of barley (Hordeum vulgare) and cultures of Lemna perpusilla have been labeled with ³²Pi and the chromatin proteins prepared and separated by acid-urea and sodium dodecyl sulfate gel electrophoresis. Under these conditions chromatin proteins became labeled and the gel radioactivity profiles which were complex indicated a probable minimum of 15 to 20 proteins phosphorylated with molecular weights ranging from 10⁴ to 10⁵. The majority of the radioactivity, 80 to 90% of the total, is found in the acidic protein fraction and this can be recovered as serine phosphate after partial acid hydrolysis.     

Newly found selenium-containing proteins in the tissues of the rat

The Se-containing proteins in 27 tissues of the rat were investigated by in vivo labeling with⁷⁵Se-selenite, separation of the tissue homogenate proteins by SDS-polyacrylamide gel electrophoresis, and determination of the labeled proteins by autoradiography. By using Se-depleted rats and a⁷⁵Se-tracer with a high specific activity, Se compounds present at only very low concentrations could be detected. Besides the 13 Se-containing proteins previously described, for which apparent molecular masses of 12, 15, 18, 20, 22, 25, 28, 34, 56, 60, 65, 70, and 75 kD have been found here, a further 15⁷⁵Se-labeled bands, with apparent molecular masses of 8, 10, 15.5, 16.5, 24, 32, 34.5, 38, 40, 41, 44, 45, 46.5, 53 and 116 kD could be distinguished. Two-dimensional separation of the kidney homogenate proteins showed that some of the Se-containing bands could be resolved into several labeled spots. Most of the newly found compounds were present in various tissues, but with some the enrichment in certain tissues suggested specific sites of action.