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B Krust

Publications and source records attributed to B Krust.

46 records · Page 3Linked to original sources
PubMed1984

Characterisation of the interferon-mediated protein kinase by polyclonal antibodies.

Interferon-treated human cells show an enhanced level of a double-stranded (ds) RNA-dependent protein kinase activity which is manifested by the phosphorylation of an endogenous 72,000 molecular weight protein (p72K kinase). By the use of murine polyclonal antibodies against this p72K kinase, here we have characterized the protein kinase activity associated with immune complexes precipitated from extracts of interferon-treated cells. Precipitation of the p72K kinase by the polyclonal antibodies results in the formation of a complex in which the kinase activity is manifested by phosphorylation of the 72K protein. This phosphorylation, however, is independent of dsRNA. Such immunoprecipitates can also phosphorylate exogenous substrates, calf thymus histones and the alpha subunit of protein initiation factor eIF2.

Animals↗
PubMed1984

Further characterization of the protein kinase activity mediated by interferon in mouse and human cells.

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous Mr = 67,000 and 72,000 proteins, respectively. Such kinase activity can be assayed after its partial purification on poly(I) X poly(C)-Sepharose. Under these experimental conditions, the apparent km of the kinase for ATP is 1.0 X 10(-6) M and 2.5 X 10(-6) M in enzyme fractions from mouse L-929 and human HeLa cells, respectively. The Mr = 67,000 and 72,000 proteins are phosphorylated by their serine and threonine residues, the ratio of which is modified in preparations from interferon-treated cells. Both of these phosphoproteins are composed of several subspecies with similar isoelectric points (pIs) in the range of 7.2 to 8.2. This heterogeneity is due to the number of phosphate groups per molecule of protein. Accordingly, the pIs of highly phosphorylated proteins are at a less basic pH (7.2 to 7.5). Furthermore, highly phosphorylated proteins show an increase in their apparent molecular weights compared to partially phosphorylated ones. This corresponds to an increase of Mr = 1,500. Partial proteolysis of the 32P-labeled Mr = 67,000 and 72,000 proteins by Staphylococcus aureus V8 protease, alpha-chymotrypsin and thrombin, indicated that these phosphoproteins differ in their polypeptide structure. Phosphorylation of the Mr = 67,000 and 72,000 proteins in enzyme fractions from control L-929 and HeLa cells is enhanced by mixing with extracts from interferon-treated heterologous cells. Proteins, Mr = 67,000 and 72,000, therefore, may serve as suitable substrates for an exogenous kinase, thus indicating that the substrate in enzyme fractions from control cells is less phosphorylated because of a low level of kinase activity.

Animals↗
PubMed1984

Interferon-mediated protein kinase activity in different fractions of mouse L-929 cells.

The interferon (IFN)-mediated protein kinase activity in extracts from mouse L-929 cells is manifested by the phosphorylation of an endogenous 67 kD molecular weight (mw) protein in the presence of double-stranded (ds) RNA. This protein kinase activity can also be assayed after partial purification on poly(I) X poly(C)-Sepharose under phosphatase-free conditions. By the use of this latter technique, here we investigated the distribution of the protein kinase activity in different cellular compartments. Most of the protein kinase activity is found in the post-ribosomal supernatant (S100) fraction, while a small portion of it is associated with the ribosomal salt wash (RSW: 0.5 M KCl eluate of ribosomal pellet) and nuclear fractions. These results are in contrast to several observations in the literature in which the protein kinase activity is thought to be associated with the ribosomal pellet. This controversy results from the conditions used for assay of the protein kinase activity. In fact, when the kinase is assayed in crude extracts supplemented with dsRNA, very little kinase activity is detectable in the S100 fraction compared to the RSW fraction. The S100 fraction contains a high level of phosphatase(s) activity which interferes with the protein kinase assay and might account for the misinterpretation observed in the literature. Some recent results have implicated a correlation between the dsRNA-dependent protein kinase responsible for the phosphorylation of the 67 kD protein and a polyamine-dependent protein kinase which phosphorylates a similar molecular weight protein, subunit of ornithine decarboxylase (Orn Dcase). Here, we show that Orn Dcase does not bind to poly(I) X poly(C)-Sepharose and polyamines do not substitute the requirement of dsRNA for the phosphorylation of the 67 kD protein.

Animals↗
PubMed1983

Phosphorylation of the alpha-chain of fibrinogen by a platelet kinase activity enhanced by interferon.

Treatment of patients with interferon or inducers of interferon results in an enhanced level of a protein kinase activity found in platelets (1,3). The kinase activity is responsible for the phosphorylation of a 70-72,000 molecular weight protein (72K protein) found in blood plasma. By the means of a technique based on the precipitation of this protein kinase system (the protein kinase and its substrate), we show here that the 72K protein is the alpha-chain of fibrinogen. During the coagulation process induced by thrombin, the 32P-labelled 72K protein is recovered in the clot. After incubation in the presence of thrombin, the 72K protein looses a small polypeptide of 2-3000 in molecular weight resulting a shift in its isoelectric point (pI) from 6.8-7.0 to 7.5. At the end of the coagulation process, the 32P-labelled 72K protein becomes undetectable since it gives rise to a covalently linked alpha-polymer of a high molecular weight. In accord with these results, the 72K protein could be precipitated by antibodies against human fibrinogen.

Antibodies↗
PubMed1983

A protein kinase system from platelet rich plasma.

Treatment of platelet rich plasma (PRP) at pH 5 results in the precipitation of a protein kinase system. The protein kinase is associated with the platelet fraction and is capable of phosphorylation of several plasma proteins. Analysis of the 32P-labeled phosphoproteins by two dimensional gel electrophoresis showed the existence of three major phosphoproteins: 72K and 80K proteins with identical isoelectric points (pI) of 6.0 and another 72K protein with a pI of 6.8-7.0. This latter 72K phosphoprotein has recently been identified as the alpha-chain of fibrinogen. The identity of the other 2 proteins remains to be shown. The activity of the protein kinase is markedly enhanced by Mn2+, it phosphorylates calf thymus histone as an exogenous substrate and is independent of cAMP or cGMP. This protein kinase activity is inhibited competitively by ADP.

Adenosine Diphosphate↗
PubMed1983

Assay of interferon-mediated protein kinase activity from plasma and tissue extracts.

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous 67,000 and 72,000 Mr proteins, respectively. Enhanced levels of such kinase activity are also detectable in the plasma of patients treated with interferon and in the plasma and tissues of interferon-treated mice. A rapid and efficient method of assay for these protein kinase activities is described. The samples are first incubated with heparin (100 units/ml), which results in the inhibition of different protein kinase activities, but not the one mediated by interferon. The latter one is then assayed after partial purification on poly(rI):(rC)-Sepharose or poly(rG)-Sepharose. The protein kinase from human and mouse cells in culture and from the different tissues of mice binds specifically to poly(rI):(rC)-Sepharose. On the other hand, the protein kinase activity from both mouse and human plasma shows a higher affinity toward poly(rG)-Sepharose. These methods are successfully applied for the determination of the interferon-mediated protein kinase activity from tissue extracts and plasma.

Animals↗
PubMed1982

Characterization of monoclonal antibody specific for human alpha interferon.

A mouse hybridoma cell line has been isolated, which secretes a monoclonal antibody specific of human leukocyte (alpha) interferon. The antibody secreted by the hybridoma belongs to the IgG1 class. It neutralizes biological activities (cellular and antiviral) of alpha interferon. Mass production of the antibody in ascitic fluid has been obtained. A convenient method of purification of the IgG from the ascitic fluid on DEAE-Trisacryl M is described.

Animals↗
PubMed1979

Bromodeoxyuridine-induced reversion of transformed characteristics in BHK21 cells: changes at the plasma membrane level.

When BHK21 cells transformed by hamster sarcoma virus are grown in the presence of 5-Bromedeoxyuridine (BUdr), several in vitro properties of the transformed cells such as morphology, adhesiveness, and alignment, revert towards a state close to that of untransformed cells. We have studied plasma membrane changes associated with this phenotypic reversion by several different biochemical methods. Reversion is accompanied by a reappearance of Fibronectin, an increase in a membrane-associated protein of M.W. 100,000 which is increased in transformed cells and a decrease in Con A-agglutinability. On the other hand, several membrane changes associated with malignant transformation namely, the increase in an integral membrane protein M.W. 177,000, the higher rate of hexose uptake, the increase in high molecular weight surface glycopeptides and, to some extent, the increase in the density of intramembranous particles, did not revert under BUdr treatment. Thus, membrane properties of transformed cells may be dissociated into two main groups by BUdr treatment. In addition, the exposure and glycosylation of a growth-regulated membrane protein M.W. 160,000 was highly sensitive to BUdr.

Agglutination↗
PubMedDate not supplied

Membrane-expressed HIV envelope glycoprotein heterodimer is a powerful inducer of cell death in uninfected CD4+ target cells.

HIV infection of CD4+ T cells in culture results in the production of virus and induction of cell killing by apoptosis. Such a cytopathic effect is observed during infection with syncytium-inducing or non-syncytium-inducing HIV isolates. Apoptosis is triggered by the interaction of the cell membrane-expressed HIV envelope glycoprotein heterodimer gp120-gp41 complex (external and transmembrane glycoprotein complex) with the CD4 receptor. Here we demonstrate an experimental model for the induction of apoptosis independent of HIV infection, using transiently transfected HeLa cells with the HIV1 env gene as effector cells and the CD4+ MOLT4-T4 T cells as target cells. Results obtained confirm that the induction of apoptosis requires the membrane expression of the two HIV env gene products, gp120 and gp41. Single amino acid point mutations of the envelope products that affect binding to the CD4 receptor or the fusion process abrogate the capacity of the gp120-gp41 complex to induce apoptosis. Interestingly, a point mutation in the V3 loop which inhibits fusion without affecting CD4 binding also results in the abrogation of apoptosis. These observations indicate that the induction of apoptosis is an intrinsic property of the cell membrane-expressed gp120-gp41 complex, and thus should be considered as one of the functions of HIV env gene products.

CD4-Positive T-Lymphocytes↗