The platelet-activating factor (PAF) was seen to potently activate mitogen-activated protein (MAP) kinase and MAP through the cloned guinea pig PAF receptor stably expressed in Chinese hamster ovary (CHO) cells. Both 42- 44-kDa kinases were activated tyrosine-phosphorylated response PAF. also triggered production of inositol phosphates release arachidonic acid inhibited cyclic AMP accumulation. Differential inhibitory effects pertussis toxin (PTX) on these signals suggested that couples both...

Abstract: A sensitive two‐site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of antigen between anti‐mouse NGF antibody IgG coated to a polystyrene tube and Fab'‐linked β‐ d ‐galactosidase (β‐ ‐galactoside hydrolase, EC 3.2.1.23). This method has following advantages: (a) procedures are simple rapid compared bioassay or radioimmunoassay; (b) Fab'‐β‐ complex is more stable than 125 I‐labeled antibody; (c) purified detectable at...

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in neck region facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) CML13 and CML14 interactors of containing multiple IQ domains, including a myosin VIII. Here, we demonstrate CaM, CML13, all four VIII isoforms. Among CMLs tested...

<i>Chara corallina</i> class XI myosin is by far the fastest molecular motor. To investigate mechanism of this fast movement, we performed a kinetic analysis recombinant motor domain <i>Chara</i> myosin. We estimated time spent in strongly bound state with actin measuring rate constants ADP dissociation from actin·motor complex and ATP-induced actin. The constant acto-motor was >2800 s^-1, at physiological ATP concentration 2200 s^-1. From these data, to be <0.82 ms. This...

The myosin superfamily comprises over 70 classes, each with multiple subclasses, and shows substantial diversity in properties such as velocity, ATPase activity, duty ratio, directionality. This functional underpins the specialized roles of various organisms, organs, cell types. Recent studies have revealed that certain myosins induce chiral curved motions actin filaments. However, this newly identified property remains largely unexplored. Here, we investigated motion vitro using Chara...

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, myosins are not characterized. Here, we report first analysis enzymatic myosin. The motor domain Arabidopsis myosin, ATM1 (ATM1-MD), plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system ATM1-MD ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin high (Kactin μM). actin-sliding...

Cytoplasmic streaming with extremely high velocity (∼70 μm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic is caused by myosin XI, it has been suggested that a XI 70 s-1, fastest measured so far, exists Chara cells. However, previously cloned corallina (CcXI) was about 20 one-third Recently, genome sequence braunii published, revealing this alga four genes. We these (CbXI-1, 2, 3, and 4) their velocities. While velocities CbXI-3 CbXI-4 motor domains (MDs) were...

Plant myosin XI acts as a motive force for cytoplasmic streaming through interacting with actin filaments within the cell. Arabidopsis thaliana (At) has 13 genes belonging to family. Previous reverse genetic approaches suggest that At XIs are partially redundant, but functionally diverse their specific tasks plant. However, tissue-specific expression and enzymatic properties of have date been poorly understood, primarily because difficulty in cloning expressing large proteins. In this study,...

According to the power stroke model of dynein deduced from electron microscopic and fluorescence resonance energy transfer studies, recovery are expected take place at two isomerization steps ATPase cycle primary site. Here, we have conducted presteady-state kinetic analyses these with single-headed motor domain Dictyostelium cytoplasmic by employing probe site tail positions. Our results show that first step proceeds quickly ( approximately 180 s(-1)), whereas second is very slow 0.2 s(-1))...

Most myosins have a positively charged loop 2 with cluster of lysine residues that bind to the negatively N-terminal segment actin. However, net charge very fast Chara myosin is zero and there no in it. In contrast, has highly 3. To elucidate role these unique surface loops its high velocity actin-activated ATPase activity, we undertaken mutational analysis using recombinant motor domain. It was found positive 3 affected V(max) K(app) actin activated while it only slightly. The velocity,...

Flowering plants express multiple actin isoforms. Previous studies suggest that individual isoforms have specific functions; however, the subcellular localization of in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative isoforms, AtACT2 AtACT7, as fluorescent-fusion proteins. By optimizing linker sequence between fluorescent protein actin, succeeded observing filaments contained these fused with green (GFP) protoplasts. Different colored...

We describe the principle and experiment of stimulated Raman scattering (SRS) microscopy, which has various advantages such as high-contrast high sensitivity. To discuss how these are realized in SRS we introduce an intuitive picture SRS, where process is viewed homodyne detection a nonlinear-optical signal by excitation pulse.

The syntheses of [2]- and [3]catenanes by olefin metathesis oxidative acetylide coupling have been studied in detail. Pseudorotaxanes that were obtained mixing crown ether ammonium salts containing two terminal reactive end-groups converted to [3]catenane. Their yields influenced not only the chain length but also concentration salts. strain energies [2]catenane responsible for formation [2]catenane.

We studied the effects of 2,3-butanedione monoxime (BDM) on cytoplasmic streaming Chara corallina and motility myosin prepared from same plant to examine whether this reagent really affects class XI myosin. It was found that BDM inhibited both at a very similar concentration range (10-100 mM). introduced directly into tonoplast-free cells also streaming. These results suggested effect exerted through not ion channels least in corallina, though high required.

Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has high ATPase activity. To estimate the energy consumed by this myosin, its concentration internodal cells of C. determined quantitative immunoblot. It considerably (200 nM) and amount ATP would exceed supplied dark respiration if all molecules were fully activated interaction with actin. These results model calculations suggested required to generate is small only one-hundredth existing enough...

We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed triple helical repeats Dictyostelium alpha-actinin, order understand its activity and intracellular function. The V(max) K(actin) actin-activated Mg(2+) ATPase were 50.7 Pi head(-1) s(-1) 30.2 microM, respectively, at 25 degrees C. could translocate actin filament maximum velocity 1.8 microm C vitro motility assay. value corresponded a 3.2 for native if...

The G146V mutation in actin is dominant lethal yeast. filaments bind cofilin only minimally, presumably because binding requires the large and small domains to twist with respect one another around hinge region containing Gly-146, inhibits that twisting motion. A number of studies have suggested force generation by myosin also undergo conformational changes. This prompted us examine effects on motility. When compared wild-type filaments, showed a 78% slower gliding velocity 70% smaller stall...

Coordination between the nucleotide-binding site and converter domain of myosin is essential for its ATP-dependent motor activities. To unveil communication pathway these two sites, we investigated contact side chains Phe-482 in relay helix Gly-680 SH1-SH2 helix. F482A myosin, which was changed to alanine with a smaller chain, not functional vivo. In vitro, did move actin filaments Mg2+-ATPase activity hardly activated by actin. Phosphate burst tryptophan fluorescence analyses, as well...

Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of thaliana (At XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs At XI-I. Unlike other biochemically studied myosins, showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity was 0.25 µm s(-1), >10 times lower than those ADP dissociation rate from...