Actin-based motor properties of native myosin VIIa

2002 ◽  
Vol 115 (2) ◽  
pp. 445-450 ◽  
Author(s):  
Igor P. Udovichenko ◽  
Daniel Gibbs ◽  
David S. Williams
Keyword(s):  
Atpase Activity ◽  
Inner Ear ◽  
Actin Filaments ◽  
Maximal Activity ◽  
Motility Assay ◽  
Calmodulin Binding ◽  
Retinal Tissue ◽  
Protein Functions ◽  
Bona Fide ◽  
Myosin Viia

Myosin VIIa has critical roles in the inner ear and the retina. To help understand how this protein functions, native myosin VIIa was tested for mechanoenzymatic properties. Myosin VIIa was immunoprecipitated from retinal tissue and found to be associated with calmodulin in a Ca2+-sensitive manner. Myosin VIIa Mg-ATPase activity was detected; in the absence of Ca2+ (i.e. with bound calmodulin), it was stimulated by f-actin with a Kcat of 4.3 s–1 and with 7 μM actin required for half-maximal activity. In a sliding filament motility assay, myosin VIIa moved actin filaments with a velocity of 190 nm s–1. These results demonstrate that myosin VIIa is a calmodulin-binding protein and a bona fide actin-based motor.

scholarly journals Unconventional Myosins in Inner-Ear Sensory Epithelia

1997 ◽  
Vol 137 (6) ◽  
pp. 1287-1307 ◽  
Author(s):  
Tama Hasson ◽  
Peter G. Gillespie ◽  
Jesus A. Garcia ◽  
Richard B. MacDonald ◽  
Yi-dong Zhao ◽  
...  
Keyword(s):  
Inner Ear ◽  
Hair Cells ◽  
Actin Filaments ◽  
Structural Integrity ◽  
Myosin Vi ◽  
Cortical Actin ◽  
Unconventional Myosin ◽  
Myosin Isozymes ◽  
Cuticular Plate ◽  
Myosin Viia

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Iβ is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Iβ is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Iβ, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Iβ probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

scholarly journals A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties

1988 ◽  
Vol 251 (3) ◽  
pp. 777-785 ◽  
Author(s):  
F Martin ◽  
J Derancourt ◽  
J P Capony ◽  
A Watrin ◽  
J C Cavadore
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein Complex ◽  
Actin Filaments ◽  
Thin Filament ◽  
Partial Amino Acid Sequence ◽  
Calmodulin Binding

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.

Functional role of ecto-ATPase activity in goldfish hepatocytes

1998 ◽  
Vol 274 (4) ◽  
pp. R1031-R1038 ◽  
Author(s):  
Pablo J. Schwarzbaum ◽  
Michael E. Frischmann ◽  
Gerhard Krumschnabel ◽  
Rolando C. Rossi ◽  
Wolfgang Wieser
Keyword(s):  
Atpase Activity ◽  
Functional Role ◽  
Maximal Activity ◽  
Hydrolysis Rate ◽  
Hyperbolic Function ◽  
Extracellular Release ◽  
Transport Atpases ◽  
Hydrolysis Of ◽  
Hepatocyte Suspension

Extracellular [γ-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus γ-32Pidue to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi ⋅ (106cells)−1 ⋅ min−1and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 μM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 μM [α-32P]ATP to the hepatocyte suspension induced the extracellular release of α-32Pi[8.2 pmol ⋅ (106cells)−1 ⋅ min−1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 μM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol ⋅ (106cells)−1 ⋅ min−1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+([Formula: see text]). This effect could be partially mimicked by adenosine 5′- O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of[Formula: see text] and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

A simple method for automatic tracking of actin filaments in the motility assay

1996 ◽  
Vol 17 (4) ◽  
pp. 497-506 ◽  
Author(s):  
Steven B. Marston ◽  
Iain D. C. Fraser ◽  
Wu Bing ◽  
Giles Roper
Keyword(s):  
Actin Filaments ◽  
Motility Assay ◽  
Simple Method ◽  
Automatic Tracking

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution

1992 ◽  
Vol 263 (2) ◽  
pp. H464-H472 ◽  
Author(s):  
H. Yamashita ◽  
S. Sugiura ◽  
T. Serizawa ◽  
T. Sugimoto ◽  
M. Iizuka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Motility Assay ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Actin Cables

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

Regulatory features of transcription in isolated mitochondria from Artemia franciscana embryos

1999 ◽  
Vol 277 (6) ◽  
pp. R1588-R1597 ◽  
Author(s):  
Brian D. Eads ◽  
Steven C. Hand
Keyword(s):  
Rna Synthesis ◽  
Maximal Activity ◽  
Artemia Franciscana ◽  
Mitochondrial Rna ◽  
Optimal Conditions ◽  
In Organello ◽  
Atp Inhibition ◽  
Bona Fide ◽  
Ph Range

Optimal conditions were developed for an in organello transcriptional run-on assay using mitochondria isolated from Artemia franciscana embryos to investigate potential regulatory features of RNA synthesis under conditions of anoxia-induced quiescence. Transcription is not dependent on oxidative phosphorylation for maximal activity when exogenous ATP is available. Bona fide transcription products, as assessed by hybridization with specific mitochondrial cDNAs from A. franciscana, are produced in an inhibitor-sensitive manner. Transcription rate measured at pH 7.9 is reduced 80% when pH is lowered to 6.3, a pH range that mimics the in vivo change seen on exposure of embryos to anoxia. The proton sensitivity of mitochondrial RNA synthesis may provide a mechanism to depress this significant energy expenditure during quiescence. The influence of nucleotide concentration on kinetics is complicated by an interdependence among nucleotide species. ATP inhibition observed at subsaturating UTP concentrations is relieved when UTP is at saturating, physiologically relevant levels. Taken together, these data suggest that local (versus nuclear mediated) control is important in dictating mitochondrial transcription during rapid modulations in gene expression, such as those observed under anoxia-induced quiescence.

scholarly journals Adaptations in human muscle sarcoplasmic reticulum to prolonged submaximal training

2003 ◽  
Vol 94 (5) ◽  
pp. 2034-2042 ◽  
Author(s):  
H. J. Green ◽  
C. S. Ballantyne ◽  
J. D. MacDougall ◽  
M. A. Tarnopolsky ◽  
J. D. Schertzer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Vastus Lateralis ◽  
Training Session ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
Protein Levels ◽  
Plasmic Reticulum ◽  
Cycle Training ◽  
The Hill

In this study, we employed single-leg submaximal cycle training, conducted over a 10-wk period, to investigate adaptations in sarcoplasmic reticulum (SR) Ca2+-regulatory proteins and processes of the vastus lateralis. During the final weeks, the untrained volunteers (age 21.4 ± 0.3 yr; means ± SE, n = 10) were exercising 5 times/wk and for 60 min/session. Analyses were performed on tissue extracted by needle biopsy ∼4 days after the last training session. Compared with the control leg, the trained leg displayed a 19% reduction ( P < 0.05) in homogenate maximal Ca2+-ATPase activity (192 ± 11 vs. 156 ± 18 μmol · g protein−1 · min−1), a 4.3% increase ( P < 0.05) in pCa50, defined as the Ca2+ concentration at half-maximal activity (6.01 ± 0.05 vs. 6.26 ± 0.07), and no change in the Hill coefficient (1.75 ± 0.15 vs. 1.76 ± 0.21). Western blot analysis using monoclonal antibodies (7E6 and A52) revealed a 13% lower ( P < 0.05) sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 in trained vs. control in the absence of differences in SERCA2a. Training also resulted in an 18% lower ( P < 0.05) SR Ca2+ uptake and a 26% lower ( P < 0.05) Ca2+ release. It is concluded that a downregulation in SR Ca2+ cycling in vastus lateralis occurs with aerobic-based training, which at least in the case of Ca2+ uptake can be explained by reduction in Ca2+-ATPase activity and SERCA1 protein levels.

scholarly journals A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

1980 ◽  
Vol 187 (2) ◽  
pp. 507-513 ◽  
Author(s):  
Douglas Mauldin ◽  
Basil D. Roufogalis
Keyword(s):  
Atpase Activity ◽  
Sucrose Gradient ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Heat Stable ◽  
Protein Activator ◽  
Human Erythrocyte Membranes

Treatment of extensively washed erythrocyte membranes with 0.1mm-EDTA decreased their Mg2+-dependent, Ca2+-stimulated ATPase [(Mg2++Ca2+)-ATPase] activity. An activator released by this treatment restored the (Mg2++Ca2+)-ATPase to its original value in a Ca2+-dependent manner. This activator was different from calmodulin, as determined by a number of criteria. It was retained on an Amicon XM-100 ultrafiltration membrane (molecular-weight cut-off 100000); it appeared in the void volume of Sephadex G-100 and G-75 columns; it was not retained on a DEAE-cellulose ion-exchange column at ionic strengths similar to those used to retain calmodulin; and it maximally activated (Mg2++Ca2+)-ATPase activity less than calmodulin and at a higher Ca2+ concentration. Like calmodulin, the activator is heat-stable. The activator fraction isolated on a 2.5–15% sucrose gradient in 0.16m-KCl showed a single band of mol.wt. 63000 and no calmodulin on 10%-polyacrylamide/sodium dodecyl sulphate gels. A trace amount of calmodulin was detected in the activator fraction by radioimmunoassay (approx. 10pg/ml of ‘ghosts’), but this amount was insufficient to account for the (Mg2++Ca2+)-ATPase activation. Furthermore, calmodulin-binding protein failed to inhibit (Mg2++Ca2+)-ATPase activity by more than 10–20% in the membrane preparations from which the activator was extracted. It was concluded that erythrocyte membranes contain a (Mg2++Ca2+)-ATPase activator that may attenuate the activation of the Ca2+-transport ATPase by calmodulin.

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

2006 ◽  
Vol 453 (6) ◽  
pp. 905-913 ◽  
Author(s):  
Takenori Miyashita ◽  
Hitoshi Tatsumi ◽  
Kimihide Hayakawa ◽  
Nozomu Mori ◽  
Masahiro Sokabe
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Inner Ear ◽  
Endolymphatic Sac

scholarly journals Drosophilia spectrin. I. Characterization of the purified protein.

1987 ◽  
Vol 105 (5) ◽  
pp. 2095-2102 ◽  
Author(s):  
R Dubreuil ◽  
T J Byers ◽  
D Branton ◽  
L S Goldstein ◽  
D P Kiehart
Keyword(s):  
Actin Binding ◽  
Contour Length ◽  
Diagnostic Features ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Vertebrate Brain ◽  
Culture Cells ◽  
Drosophila Protein ◽  
Bona Fide

We purified a protein from Drosophila S3 tissue culture cells that has many of the diagnostic features of spectrin from vertebrate organisms: (a) The protein consists of two equimolar subunits (Mr = 234 and 226 kD) that can be reversibly cross-linked into a complex composed of equal amounts of the two subunits. (b) Electron microscopy of the native molecule reveals two intertwined, elongated strands with a contour length of 180 nm. (c) Antibodies directed against vertebrate spectrin react with the Drosophila protein and, similarly, antibodies to the Drosophila protein react with vertebrate spectrins. One monoclonal antibody has been found to react with both of the Drosophila subunits and with both subunits of vertebrate brain spectrin. (d) The Drosophila protein exhibits both actin-binding and calcium-dependent calmodulin-binding activities. Based on the above criteria, this protein appears to be a bona fide member of the spectrin family of proteins.

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