Fumi Morita

Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle1

The cDNA encoding a phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147‐residue polypeptide, which we termed CPI17, a 17‐kDa PKC‐potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non‐muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.

Interaction of heavy meromyosin with substrate II. Rate of the formation of ATP-induced ultraviolet difference spectrum of heavy meromyosin measured by stopped-flow method

Abstract 1. 1. The rate of formation of the ultraviolet difference spectrum of heavy meromyosin, induced by ATP, was measured by the stopped-flow method. The initial velocity of formation was proportional to the initial concentration of ATP, indicating a second-order reaction. The second-order rate constant decreased with decrease of MgCl 2 and with increase of KCl concentrations, but did not depend on the kind of divalent cation (Mg 2+ or Ca 2+ ) in the medium. 2. 2. The maximum difference absorbance Δ ; A max attained after addition of ATP was obtained as a function of initial concentration of ATP. A strong binding of ATP to heavy meromyosin was suggested in the presence of MgCl 2 . The minimum ATP concentration giving the maximum Δ ; A max was 2-fold the protein concentration. In the presence of CaCl 2 , the binding was not as strong. 3. 3. Decay of the difference spectrum was measured. In the presence of MgCl 2 , k s calculated using Chance s equation agreed well with the maximum velocity of the steady-state ATPase. In the presence of CaCl 2 , k s was 3 times larger than the maximum velocity. 4. 4. It is inferred that the difference spectrum accompanies only the Michaelis-Menten complex formed at one of the two sites in heavy meromyosin.

Interaction of heavy meromyosin with substrate III. Difference spectrum of ultraviolet absorption in subfragment 1 induced by ATP or ADP

Abstract 1. 1. The ultraviolet absorption difference spectrum of Subfragment 1 induced by ATP was measured in the presence of MgCl 2 . The shape of the difference spectrum was the same as that of heavy meromyosin. The difference molar extinction coefficient at the major peak, ΔE 289 m μ , was 2200 ± 400 M −1 ·cm −1 , which was 44% of that of heavy meromyosin. It decreased with time especially near 290 mμ and reached an identical shape as that induced by ADP. 2. 2. The difference spectra of Subfragment 1 induced by the addition of ADP and of heavy meromyosin had identical forms. ΔE 288 m μ was 1300 ± 400 M −1 ·cm −1 , which was 52% of the value of heavy meromyosin. 3. 3. The dependence on the concentration of the difference absorbance induced by ADP was measured at 288 mμ. A strong binding of ADP to Subfragment 1 was suggested. The minimum ADP concentration needed to give the maximum change in the difference absorbance was 1.2 times the concentration of Subfragment 1. 4. 4. Both difference spectra due to ATP and ADP were not dependent on the KCl concentration. 5. 5. Decay of the difference spectrum of Subfragment 1 induced by ATP was measured. The value of k s obtained from Chances equation agreed with the maximum velocity of steady-state ATPase.

Multiple effects of CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity on HIV-1 gp120 functions

The interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity has been studied. Conformational changes in gp120, which could affect its interaction with CD4 and its shedding from virions, were detected by fluorescence spectrum analysis of tryptophan residues after addition of peptide representative of the CD4 CDR3-related region, but not the CD4 CDR2-related region. Interestingly, the addition of scrambled peptide, S1 (with altered amino acid sequence compared with the native CDR3-related peptide but unaltered overall composition), which we recently showed to have stronger anti-HIV-1 activity than the original CDR3-related peptide, had no effects on the conformational change in gp120 or on its interaction with CD4 and its shedding from HIV-1 virions. However, all of the CDR3-related peptides, including S1, showed blocking effects on the binding of antibodies against gp120 V3 loop and C-terminus regions. Thus, we concluded that there were at least two separable activities of the CDR3-related peptides in anti-HIV-1 activity, i.e. induction of conformational changes in gp120, which could affect its binding to CD4 and to gp41 (as observed in native CDR3-related peptides), and inactivation of V3 loop and C-terminus regions in gp120 (as observed in all of the CDR3-related peptides, including S1).

An Actin-Binding Site on Myosin

In order to understand the molecular mechanism of muscle movement or cell motility, identification and characterization of actin-binding sites on myosin head and myosin-binding sites on actin molecules are necessary. Elucidation of the correlation between the binding sites and the myosin ATPase reaction is also essential. We will discuss here an actin-binding site located around the reactive Cys(SH1)(705) on the myosin heavy chain and its role in the actomyosin ATPase reaction.

Actin-binding site of pig cardiac myosin.

An actin-binding site is also present in the tryptic 20 kDa peptide fragment of the subfragment-1 heavy chain of pig cardiac myosin. As previously reported for skeletal myosin (Katoh, T., Katoh, H., and Morita, F. (1985) J. Biol. Chem. 260, 6723-6727), the site was further narrowed down to the 10 kDa peptide containing the reactive SH1 and SH2 groups. Thus it appears that the actin-binding site around the two thiols found in skeletal myosin is common to different types of myosin.

Different modes of interaction of two peptide fragments from subdomain 4 of rabbit skeletal muscle actin with actin protomers

Previously, we reported that the 2.6 kDa peptide fragment extending from Arg-177 to Tyr-198 in rabbit skeletal muscle actin bound to actin itself and inhibited its polymerization, while the 9.1 kDa peptide extending from Ser-199 to Tyr-279 in actin did not. The 2.6 kDa segment of actin was reported to contain one of the important actin-actin contacts (Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408). In this paper, we show additional evidence that the rate of salt-induced increase in the fluorescence of pyrene-labeled actin was decreased in the presence of the 2.6 kDa peptide. Conventional actin filaments were only scarcely observed in the presence of the 2.6 kDa peptide under an electron microscope with a steady state of fluorescence increase. Furthermore, the 2.6 kDa peptide was found to sever F-actin into short filament fragments. The 9.1 kDa peptide, on the other hand, neither inhibited the fluorescence increment of pyrene-actin nor severed actin filaments. However, the 9.1 kDa peptide was found to increase the viscosity and fluorescence intensity of pyrene-G-actin and to form short actin filaments in the absence of salts. Contact sites in the 9.1 kDa segment in actin may have a different mode of interaction with adjacent actin promoters in actin filaments from that of the 2.6 kDa segment.