Search PubMed⌕ Search

Biomedical subjects

J C Bulinski

Publications and source records attributed to J C Bulinski.

64 records · Page 4Linked to original sources
PubMed1983

Peptide antibody specific for the amino terminus of skeletal muscle alpha-actin.

The NH2-terminal peptide of skeletal muscle alpha-actin (S alpha N peptide), which contains a primary sequence unique to this actin isozyme, was used to prepare an isozyme-specific peptide antibody. S alpha N peptide was purified from chicken breast muscle actin by preparative reverse-phase HPLC and was coupled to hemocyanin. This complex was used to immunize rabbits in order to elicit actin antibodies specific for the skeletal muscle alpha-actin isozyme. The antibody obtained, called S alpha N antibody, was reactive with S alpha N peptide and with skeletal muscle alpha-actin as well as with cardiac muscle alpha-actin. S alpha N antibody did not react with either of the actin isozymes present in smooth muscle (smooth muscle alpha and gamma) or in brain (nonmuscle beta and gamma). S alpha N antibody was used to detect muscle-specific actin in differentiating mouse and human myoblasts by using immunoblots of myoblast extracts and immunofluorescent staining of fixed cells.

Actins↗
PubMed1981

Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins.

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca(2+) sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas microtubules are completely stable at Ca(2+) concentrations below 1 muM, Ca(2+) at greater than 1-4 muM induces microtubule disassembly that begins in the cell periphery and proceeds towards the cell center. At concentrations of up to 500 muM, both the pattern and time course of disassembly are not markedly altered, suggesting that, within this concentration range, Ca(2+) effects are catalytic rather than stoichiometric. Higher (millimolar) Ca(2+) concentration results in rapid destruction of microtubules. Of other divalent cations, only Sr(2+) has a slight depolymerizing effect, whereas millimolar Ba(2+), Mg(2+), or Mn(2+) is ineffective. Disassembly induced by micromolar Ca(2+) is inhibited by pharmacological agents known to bind to calmodulin and inhibit its function, suggesting that calmodulin mediates Ca(2+) effects. Both the addition of exogenous brain microtubule-associated proteins (MAPs) after lysis and the retention of endogenous cellular MAPs normally extracted during the lysis step stabilize microtubules against the depolymerizing effect of micromolar Ca(2+). The results indicate that, in this model system, microtubules are sensitive to physiological Ca(2+) concentrations and that this sensitivity may be conferred by calmodulin associated with the microtubules. MAPs appear to have a modulating effect on microtubular Ca(2+) sensitivity and thus may function as a discriminating factor in cellular functions performed by calmodulin. It is hypothesized that Ca(2+)-stimulated microtubule disassembly depends on the relative amount of MAPs.

Animals↗
PubMed1981

Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates.

Results from ultrastructural immunocytochemistry on glutaraldehyde-fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules.

Alkaloids↗
PubMed1980

Microtubule-associated proteins from cultured HeLa cells. Analysis of molecular properties and effects on microtubule polymerization.

We have attempted a biochemical characterization of the microtubule-associated proteins (MAPs) of cultured HeLa cells. The HeLa MAPs consist of a group of three polypeptides of 200,000 to 220,000 molecular weight (the 210K MAP) and a protein of 125,000 molecular weight (the 125K MAP). The solution properties of the HeLa MAPs were examined using molecular sieve chromatography and sucrose gradient sedimentation. With both analytical procedures, the 125K and 210K MAPs behaved independently of one another. The effects of each of the MAPs on microtubule polymerization were also studied. Both the 125K and 210K MAPs stimulated the polymerization of pure tubulin. The effect of high levels of MAPs on microtubule polymerization was also examined. Increasing the concentration of MAPs at a constant tubulin concentration increased both the rate and extent of microtubule polymerization. The 125K and 210K MAPs showed independent behavior with respect to binding to microtubules. The 210K MAP saturated its binding sites at a level of 14.0% (210K MAP:tubulin in polymer, w/w), while the 125K MAP showed no saturation even at a level of 18.3%. These results demonstrate that the 125K and 210K MAPs are distinct molecular species, differing in their solution properties and their binding to microtubules. The 210K MAP showed some similarities and some differences when compared to the porcine brain high molecular weight MAP. The HeLa MAPs, although showing some properties similar to brain MAPs, are nevertheless distinctive in several respects and may best be considered as separate though possibly related species.

Centrifugation, Density Gradient↗
PubMed1980

Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo.

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross-reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross-reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.

Fluorescent Antibody Technique↗
PubMed1980

Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates.

Antisera prepared against a 210,000 mol wt microtubule-associated protein (210k MAP) isolated from the human cell line, HeLa, were used to survey a variety of cells and tissues for the presence of immunologically related proteins. The antisera were employed to test extracts of the cells and tissues, using a sensitive indirect immunofluorescence technique applied to polyacrylamide gels. Cross-reactive material of 210,000 mol wt was found in 10 kinds of cells and tissues derived from humans and four lines of cells from monkeys. Indirect immunofluorescent staining was also carried out on fixed cells and showed that the cross-reactive material was localized to interphase and mitotic microtubules as assayed in nine human and seven monkey cell lines. No protein that cross-reacted with 210k MAP antisera was detected in cells and tissues derived from two rodents, an ungulate, a marsupial, or a chicken. Therefore, the 210k MAP isolated from HeLa cells is present in a wide variety of cells and tissues of humans and other primates but is antigenically distinct from MAPs present in lower organisms.

Animals↗
PubMed1979

Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins.

Microtubule protein from HeLa cell extracts was purified by multiple cycles of polymerization and depolymerization in the absence of glycerol or other exogenous polymerization-stimulatory agents. Approximately 4-5% of the extract protein was tubulin, of which more than one-half was competent to participate in polymerization-depolymerization cycles. The purified HeLa microtubule protein preparations contained 95% tubulin after the second cycle of polymerization and depolymerization. Additional protein species bound specifically to and copurified quantitatively with microtubules throughout at least four cycles of polymerization and depolymerization. These microtubule-associated proteins (MAPs) were separated from tubulin by DEAE column chromatography. When added to purified brain or HeLa tubulin, these MAPs stimulated the polymerization of microtubules as assayed by electron microscopy and a quantitative sedimentation assay. The most prominent HeLa MAPs had molecular weights of approximately 210,000 and 120,000.

HeLa Cells↗