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M C Wagner

Publications and source records attributed to M C Wagner.

13 recordsLinked to original sources
PubMed1992

Purification and characterization of a mammalian myosin I.

Myosin I, an actin-dependent force-generating enzyme, has been purified from three mammalian sources: bovine adrenal medulla, adrenal cortex, and brain. The purification procedure includes extraction of tissue with ATP at low ionic strength and coprecipitation with actin, followed by gel filtration on Sepharose 4B, anion-exchange chromatography on Q Sepharose, and affinity chromatography on ATP-agarose. Mammalian myosin I molecules are composed of a heavy chain of 116 kDa and multiple low molecular weight polypeptides identified as calmodulin. The structural and enzymatic properties of adrenal medulla myosin I were further characterized. This enzyme exhibits high K+,EDTA- and Ca(2+)-ATPase specific activities (about 0.2 mumol.min-1 per mg of protein), whereas the Mg(2+)-ATPase activity is very low (1-3 nmol.min-1.mg-1). The Mg(2+)-ATPase of medulla myosin I is activated by F-actin in a Ca(2+)-dependent manner: activity is stimulated 40-fold in the presence of EGTA and 90-fold in the presence of 10 microM Ca2+. Two structural domains of the myosin I heavy chain were identified. A 74-kDa chymotryptic fragment contains the catalytic site, while a 36-kDa polypeptide contains the calmodulin-binding sites. These results indicate that mammalian myosin I is more closely related to myosin I from the avian intestinal brush border than to the enzymes isolated from the protozoans Acanthamoeba and Dictyostelium.

Actins↗
PubMed1992

Tissue distribution and subcellular localization of mammalian myosin I.

Myosin I, a nonfilamentous single-headed actin-activated ATPase, has recently been purified from mammalian tissue (Barylko, B., M. C. Wagner, O. Reizes, and J. P. Albanesi. 1992. Proc. Natl. Acad. Sci. USA. 89:490-494). To investigate the distribution of this enzyme in cells and tissues mAbs were generated against myosin I purified from bovine adrenal gland. Eight antibodies were characterized, five of them (M4-M8) recognize epitope(s) on the catalytic "head" portion of myosin I while the other three (M1-M3) react with the "tail" domain. Immunoblot analysis using antiadrenal myosin I antibody M2 demonstrates the widespread distribution of the enzyme in mammalian tissues. Myosin I was immunolocalized in several cell types including bovine kidney (MDBK), rat kidney (NRK), rat brain, rat phaeochromocytoma (PC12), fibroblast (Swiss 3T3), and CHO cells. In all cases, myosin I was concentrated at the cell periphery. The most intense labeling was observed in regions of the cell usually associated with motile activity (i.e., filopodia, lamellipodia and growth cones). These results are consistent with earlier observations on protozoan myosin I that suggest a motile role for the enzyme at the plasma membrane.

Animals↗
PubMed1991

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.

Algorithms↗
PubMed1991

Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity.

The protocols described here have proved to be an effective method for preparation of kinesin suitable for biochemical, biophysical, and immunological analyses. Beginning with a 1.2-liter cytosolic extract of bovine brain containing approximately 24 g of protein, 2 mg of approximately 95% pure kinesin can be obtained within 2 days. There are four major enrichment steps, as summarized in Fig. 6 and Table I. Based on quantitative SDS-PAGE, we estimate that these steps result in a purification of more than 300-fold. The ATPase activity in the presence of microtubules is substantial, and the kinetic properties are consistent with cellular levels of ATP (Km approximately 0.2 mM) and microtubules (apparent Km for activation approximately 1.9 microM) in the axon. Minor modifications should allow the procedure to be enlarged or reduced in scale, or adapted to the brains of other vertebrate species. The availability of such procedures will greatly facilitate future studies of the cell and molecular biology of kinesin.

Adenosine Triphosphatases↗
PubMed1989

Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport.

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.

Adenosine Triphosphatases↗
PubMed1989

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration.

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.

Adenosine Triphosphatases↗
PubMed1989

Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties.

Determination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5'-adenylylimidodiphosphate (AMP-PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule-activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmax of 0.31 mumol/min/mg in the presence of microtubules, with a Km for ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation-ATP complexes indicated that kinesin is a microtubule-stimulated Mg-ATPase, but kinesin is able to hydrolyze Ca-ATP, Mn-ATP, and Co-ATP as well as Mg-ATP in the presence of microtubules. In the absence of microtubules, Ca-ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP-PNP. Other inhibitors of kinesin include N-ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.

Adenylyl Imidodiphosphate↗
PubMed1989

Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells.

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.

Adenosine Triphosphatases↗
PubMed1988

Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide.

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.

Adenosine Triphosphate↗