PubMed Health⌕ Search

Biomedical subjects

R Ishikawa

Publications and source records attributed to R Ishikawa.

At least 19 recordsLinked to original sources
PubMed1992

A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin.

The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables.

Actins↗
PubMed1992

A novel regulatory protein that affects the functions of caldesmon and myosin light chain kinase.

A caldesmon (CaD)-binding protein of about 65 kDa (by SDS-PAGE) was purified from smooth muscle of chicken gizzard. The 65-kDa protein prevented the inhibitory effect of CaD on the ATP-dependent interaction between actin and myosin. Unlike the case with calmodulin (CaM), Ca2+ was not required for this effect. As reported in the preceding communication, myosin light chain kinase (MLCK), another well characterized protein that binds CaM, has CaD-like activity that modulates the interaction by binding to actin. The 65-kDa protein was also effective in relieving the modulation, while leaving unaffected the kinase activity that phosphorylates the light chain of smooth muscle myosin.

Actins↗
PubMed1992

The binding of nonmuscle caldesmon from brain to microtubules. Regulations by Ca(2+)-calmodulin and cdc2 kinase.

Nonmuscle caldesmon from bovine brain bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with values of Ka 4.5 x 10(5) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. The phosphorylation of caldesmon by cdc2 kinase also eliminated the microtubule-binding activity. These results suggest that caldesmon may play a physiological role in the functions of microtubules.

Animals↗
PubMed1992

Injury-induced reduction of acidic fibroblast growth factor levels in the distal parts of rat sciatic nerve.

Acidic fibroblast growth factor (aFGF) level in sciatic nerve after lesioning was measured by enzyme immunoassay to determine if aFGF functions as a neurotrophic factor like nerve growth factor (NGF). Whereas the NGF level increased in distal segments, the aFGF level there decreased after transection or crushing and recovered to the original level by 10 weeks after crushing. The amount of aFGF mRNA in the sciatic nerve was extremely low to supply the high level of protein found in the sciatic nerve. Sympathetic ganglia, dorsal root ganglia, and spinal cord, which contain neuronal cell bodies extending their axons into the sciatic nerve, showed a greater or similar level of aFGF as sciatic nerve. These results imply that aFGF is synthesized in neuronal cell bodies and distributed anterogradely into their axons. Difference of injury-induced changes in levels between aFGF and NGF suggests distinct mechanisms of the effects elicited from these factors on regeneration of the sciatic nerve.

Animals↗
PubMed1992

Characterization of smooth muscle caldesmon as a microtubule-associated protein.

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.

Animals↗
PubMed1992

Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth.

Albino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNA(Glu) gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered that trnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.

Base Sequence↗
PubMed1992

Regulation by Ca(2+)-calmodulin of the actin-bundling activity of Physarum 210-kDa protein.

From the plasmodia of a lower eukaryote, Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca2+. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 +/- 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 A and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations of Physarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.

Actins↗
PubMed1991

Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon.

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.

Actins↗
PubMed1991

Appearance of nerve growth factor and acidic fibroblast growth factor with different time courses in the cavity-lesioned cortex of the rat brain.

Time-dependent changes in both nerve growth factor (NGF) and acidic fibroblast growth factor (aFGF) levels in the rat brain after cortical cavity lesioning were examined, by using sensitive enzyme immunoassays (EIA) specific for each factor. In the cavity fluid, the NGF level increased rapidly and temporarily with a sharp peak 16 h after lesioning. A relatively high level was sustained during the next 3-6 days. Contrary to NGF, aFGF was first detectable only 10 days after lesioning, and its level increased gradually until 30 days. These results suggest that NGF and aFGF would play some roles for neuronal repair in different ways.

Animals↗
PubMed1991

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum.

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.

Calcium↗
PubMed1991

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon.

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.

Actins↗
PubMed1991

Characterization of calcium-binding light chain as a Ca(2+)-receptive subunit of Physarum myosin.

Physarum myosin is uniquely under an inhibitory Ca(2+)-regulation in the ATP-dependent interaction with actin [Kohama (1990) Trends Pharmacol. Sci. 11, 433-435, for review]. Calcium-binding light chain (CaLc) has been suggested to be of primary importance to the control from its amino acid sequence [Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313]. To provide a biochemical basis for this suggestion, the Ca-binding capacity of CaLc and its Kd for Ca2+ were measured. The Ca-binding properties of CaLc allowed those of Physarum myosin to be explained in terms of CaLc. However, the mode of Ca(2+)-regulation by CaLc differs according to the enzyme upon which Ca-sensitivity is confered by CaLc, i.e., CaLc activated bovine phosphodiesterase activity and inhibited Physarum myosin ATPase activity, with the same Kd in microM levels. Thus, CaLc appears to work as a mere Ca-receptive subunit in Physarum myosin, with the secret of the inhibition lying in other subunits. CaLc was also shown to belong to a family of alkali light chains (AlLc) by allowing it to bind skeletal myosin as a substitute for its AlLc. Therefore, present study is the first biochemical indication that the AlLc family is involved in regulating the myosin function.

Actins↗
PubMed1991

Developmental changes in distribution of acidic fibroblast growth factor in rat brain evaluated by a sensitive two-site enzyme immunoassay.

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.

Aging↗
PubMed1991

A myosin-like protein from smooth muscle.

A protein was purified from chicken gizzard smooth muscle. It bound ATP and actin. Actin activated the Mg2(+)-ATPase activity of this protein. The Ca2(+)-ATPase activity was lower than K(+)-EDTA ATPase activity. Thus, it appears that this protein is akin to myosin I rather than to conventional myosin. However, ATPase activities of the protein were much lower than those of myosin I. A protein cofactor, such as protein kinase, which would enhance these activities remains to be purified from the smooth muscle.

Actins↗
PubMed1991

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.

Animals↗