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A Koffer

Publications and source records attributed to A Koffer.

28 records · Page 2Linked to original sources
PubMed1988

Identification of two species of actin depolymerizing factor in cultures of BHK cells.

High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

Actin Depolymerizing Factors↗
PubMed1987

Activation of myosin ATPase by actin isolated from cultured BHK cells and the effect of gelsolin.

Activation of skeletal muscle myosin and myosin subfragment-1 (S1) by actin purified from the cytoplasm of cultured BHK cells was studied using the fluorescence of pyrene-labelled BHK F-actin and its quenching by S1 and by an enzyme-linked ATPase assay. At non-saturating concentrations, both muscle and BHK actin activated skeletal muscle myosin to the same degree: at 30 degrees C and an ionic strength of 108 mM, 1 microM actin approximately doubled the ATPase of myosin or of S1. The association between BHK actin and S1 was also followed in a fluorescence stop flow: the rate of ATP binding monitored by the loss of light scattering upon dissociation of actin was again the same for BHK and muscle actin. The similarity of activation of myosin ATPase by BHK and muscle actin at low actin concentrations (i.e. the similarity of Vmax/Km) suggests that both Vmax and Km are similar for the two types of actin. The effect of varying filament length on actin activation of myosin ATPase was examined using pig plasma or BHK gelsolin to regulate the length. For both types of actin, maximum enhancement of the actomyosin ATPase activity was observed at an actin/gelsolin ratio of about 30:1, whereas inhibition was observed at lower ratios. Both activation and inhibition of actomyosin ATPase were apparent in the absence or presence of calcium; differences were observed only in the extent and the time course of the effect.

Actins↗
PubMed1987

Isolation and characterization of actin from cultured BHK cells.

Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg2+ ions (0.2 mM CaCl2, 85 mM KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca2+ ions for BHK actin than for muscle actin (12 mM and 8 mM respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.

Actins↗
PubMed1985

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells.

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1985

Actin-regulating activities in cultured BHK cells.

A large proportion (60-85%) of the actin present in lysates of cultured baby hamster kidney (BHK) cells is monomeric at low temperature. All this G-actin is accounted for in the fractions obtained by high-speed centrifugation of the lysate in the presence or absence of non-ionic detergent; 98 (+/- 7)% of its reacts as monomer in the deoxyribonuclease assay. The fractions nevertheless possess actin polymerization nucleating activity, as assayed by the fluorescence of extraneous pyrene-conjugated actin, reflecting the presence of capping proteins and their complexes with oligomeric actin, which dissociate during the deoxyribonuclease assay. Despite the enhanced nucleation, the total proportion of actin polymerized at equilibrium is reduced, relative to that of actin in the absence of cell extract, presumably because of the presence of 'barbed'-end capping protein(s). With increasing concentrations of the supernatant material, the proportion of polymerized actin was progressively reduced and the presence of inhibitory activity against polymerization became apparent. On addition of micromolar concentrations of free calcium, nucleation by the supernatant fractions is greatly accelerated, but the extent of polymerization undergoes a further reduction, due to the elevated critical monomer concentration resulting from capping. Inhibition of polymerization by the supernatant fraction in the presence of calcium is much greater than by saturating concentrations of cytochalasin E. These observations are most simply interpreted as showing that the cytoplasm additionally contains a 'pointed'-end capping protein. The plasma membranes were found to have nucleating activity largely residing in the Triton-insoluble fractions. Actin cross-linking activity was assayed by adding small proportions of either the supernatant or membrane fractions to a large excess of F-actin and analysing the pellet resulting from a low-speed centrifugation. Supernatant fraction, containing Triton-soluble membrane-derived material, possessed much greater cross-linking capacity than the membrane-free supernatant prepared in the absence of detergent. The membrane fractions had practically no cross-linking activity on their own. Nevertheless, a non-ionic detergent extract of the membrane enhanced the cross-linking activity of the supernatant, indicating the participation of more than one species in a cross-linking process.

Actin Cytoskeleton↗
PubMed1982

Comparative studies of nuclear phosphoproteins from four different muscle tissues and liver.

1. Nuclei isolated from red and white skeletal muscle, heart, uterus and liver of rabbit were incubated with [lambda-32P]ATP and the incorporation of 32P into nuclear protein was examined. 2. Liver nuclear proteins were phosphorylated to a much greater extent than those of muscle. Particularly low levels of phosphorylation were observed in white greater extent than those of muscle. Particularly low levels of phosphorylation were observed in white muscle nuclei. 3. Most of the phosphoproteins were common to all the tissues but significant quantitative differences and a limited number of qualitative differences were evident, particularly between liver and the muscle tissues. 4. Many phosphoproteins detected were minor components of the total nuclear protein.

Adenosine Triphosphate↗