Myosin V

Calcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell.

Download Full-text

Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)86939-3 ◽

1990 ◽

Vol 265 (18) ◽

pp. 10250-10254 ◽

Cited By ~ 3

Author(s):

D M McCord ◽

D M Monroe ◽

K J Smith ◽

H R Roberts

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Conformational Change ◽

Calcium Binding ◽

Factor Ix ◽

Epidermal Growth Factor Domain ◽

High Affinity ◽

Functional Defect ◽

Epidermal Growth

Download Full-text

Iodination of Calsequestrin in the Sarcoplasmic Reticulum of Rabbit Skeletal Muscle: A Re-Examination

Australian Journal of Biological Sciences ◽

10.1071/bi9790177 ◽

1979 ◽

Vol 32 (2) ◽

pp. 177 ◽

Cited By ~ 3

Author(s):

Ronald K Tume

Keyword(s):

Skeletal Muscle ◽

Molecular Weight ◽

Sarcoplasmic Reticulum ◽

Atpase Activity ◽

Calcium Binding ◽

Local Environment ◽

Maximal Inhibition ◽

High Affinity ◽

Tris Maleate ◽

Sepharose 4B

The exposed proteins of sarcoplasmic reticulum (SR) vesicles from skeletal muscle were iodinated with the use of Sepharose 4B-bound lactoperoxidase, so that the location of the proteins in the membrane could be determined. It was found that the pattern of protein labelling could be modified simply by changing the constituents of the incubation media. This implies that the position or configuration of a particular protein in the membrane can be altered by the local environment. When the reaction was performed in the presence of 25 mM tris-maleate, pH 7 �0, alone, the Ca2+ pump ATPase (molecular weight 105000) and calsequestrin (47000) were both heavily labelled, suggesting they are at least partially exposed on the outer surface of the membrane. By contrast the high affinity calcium-binding protein (55000) was not labelled. However, when the vesicles were iodinated under conditions that were suitable for ATPase activity and Ca2+ accumulation, namely in the presence of 25 mM tris-maleate, pH 7 �0, 5 mM ATP, 5 mM Mg2+ and 0�05 mM Ca2+, a different pattern of labelling was obtained. No labelling of calsequestrin was observed whereas the extent of labelling of the Ca2+ pump ATPase remained about the same. The inclusion of anyone of the additives mentioned was effective in inhibiting the iodination of calsequestrin in the SR vesicle. When added alone, Ca2+ was more effective than Mg2+ in preventing labelling of calsequestrin. Half-maximal inhibition was observed at concentrations of approximately 0�05 mM Ca 2+ and 0�2-0�3 mM Mg2+ . Although much reduced, significant labelling of calsequestrin was observed even in the presence of 5 mM ATP. Investigations with partially purified calsequestrin revealed that the iodination of calsequestrin was the same in both the presence and absence of 1 mM Ca2 +. Therefore the reduction in label observed in intact SR vesicles probably represents a change in the location of calsequestrin within the membrane, rather than inhibition by Ca2+ of the iodination sites of the protein itself.

Download Full-text

Neuronal and Extraneuronal Uptake of 3H-Norepinephrine in the Heart of the Active Bat: An Electron Microscopic Radioautographic Study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073052 ◽

1973 ◽

Vol 31 ◽

pp. 522-523

Author(s):

Martin Hagopian ◽

Michael D. Gershon ◽

Eladio A. Nunez

Keyword(s):

Smooth Muscle ◽

Monoamine Oxidase ◽

Vascular Smooth Muscle ◽

Cardiac Muscle ◽

Maximum Rate ◽

External Medium ◽

Extraneuronal Uptake ◽

Electron Microscopic ◽

Cardiac Tissues ◽

High Affinity

The ability of cardiac tissues to take up norepinephrine from an external medium is well known. Two mechanisms, called Uptake and Uptake respectively by Iversen have been differentiated. Uptake is a high affinity system associated with adrenergic neuronal elements. Uptake is a low affinity system, with a higher maximum rate than that of Uptake. Uptake has been associated with extraneuronal tissues such as cardiac muscle, fibroblasts or vascular smooth muscle. At low perfusion concentrations of norepinephrine most of the amine taken up by Uptake is metabolized. In order to study the localization of sites of norepinephrine storage following its uptake in the active bat heart, tritiated norepinephrine (2.5 mCi; 0.064 mg) was given intravenously to 2 bats. Monoamine oxidase had been inhibited with pheniprazine (10 mg/kg) one hour previously to decrease metabolism of norepinephrine.

Download Full-text

Evidence for a Ca2+-Specific Conformational Change in Avian Thymic Hormone, a High-Affinity β-Parvalbumin

Biochemistry ◽

10.1021/bi900029j ◽

2009 ◽

Vol 48 (18) ◽

pp. 3936-3945 ◽

Cited By ~ 9

Author(s):

Anmin Tan ◽

Michael T. Henzl

Keyword(s):

Conformational Change ◽

Thymic Hormone ◽

High Affinity

Download Full-text

Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423520112 ◽

2015 ◽

Vol 112 (22) ◽

pp. 6991-6996 ◽

Cited By ~ 18

Author(s):

Takashi Suzuki ◽

Miho Suzuki ◽

Shinji Ogino ◽

Ryo Umemoto ◽

Noritaka Nishida ◽

...

Keyword(s):

Shear Stress ◽

Conformational Change ◽

Mechanical Force ◽

Binding Domain ◽

Terminal Region ◽

Hematopoietic Progenitor Cell ◽

High Shear ◽

Cell Rolling ◽

High Affinity ◽

C Terminus

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

Download Full-text