Biomedical subjects

R C Lu

Publications and source records attributed to R C Lu.

At least 19 recordsLinked to original sources

PubMed2000

The major myosin-binding site of caldesmon resides near its N-terminal extreme.

The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.

Amino Acid Sequence↗

PubMed1998

Phosphorylation changes the spatial relationship between Glu124-Arg143 and Cys18 and Cys165 of the regulatory light chain in smooth muscle myosin.

Regulatory light chain (RLC) mutants, RLC-C18 and RLC-C165, containing a single cysteine at positions 18 and 165 near the N and C terminus, respectively, were each labeled with benzophenone 4-iodoacetamide and exchanged into myosin in their phosphorylated or unphosphorylated forms and then photolyzed. SDS-PAGE showed that, for RLC-C18, the intrachain photo-cross-linking in myosin was inhibited by phosphorylation. For myosin containing RLC-C165, the yield of one intrachain cross-linked band decreased significantly whereas the other was unaffected by phosphorylation. Peptide mapping in conjunction with mass spectrometry showed that Cys165 was cross-linked to site(s) within Ala17-Lys34 independent of the phosphorylation of Ser19. This clearly demonstrates that the proximity between the N- and C-terminal regions of RLC is not affected by phosphorylation. In addition, Cys165 could also be cross-linked to the region of Phe133-Arg143; however, this type of cross-linking was inhibited in the phosphorylated state. For RLC-C18, the cross-linking took place with the region of Glu124-Arg132 or Phe133-Arg143, also only in the unphosphorylated state. Thus, phosphorylation changes the spatial relationship between the region of Glu124-Arg143 and Cys18 and Cys165. In scallop myosin, the region corresponding to Glu124-Arg143 is located at the interfaces between RLC and the essential light chain as well as the heavy chain [Xie, X. , et al. (1994) Nature 368, 306-312]. In light of that work, our results suggest that the region of Glu124-Arg143 is involved in the phosphorylation-dependent signaling and the change in its spatial relationship with respect to the N and C termini of RLC may underlie the activation of the smooth muscle myosin.

Amino Acid Sequence↗

PubMed1991

A long helix from the central region of smooth muscle caldesmon.

The central region of smooth muscle caldesmon is predicted to form alpha-helices on the basis of its primary structure. We have isolated a fragment (CT54) that contains this region. The hydrodynamic properties and the electron microscopic images suggest that CT54 is an elongated (35 nm), monomeric molecule. The circular dichroic spectrum yields an overall alpha-helical content of 55-58%. These results are consistent with the model that the middle portion of CT54 forms a long stretch of single-stranded alpha-helix. Such a structure, if it in fact exists, is thought to be stabilized by numerous salt bridges between charged residues at positions i and i + 4. The structural characteristics of this fragment not only represent an unusual protein configuration but also provide information about the functional role of caldesmon in smooth muscle contraction.

Amino Acid Sequence↗

PubMed1991

Localization of the calmodulin- and the actin-binding sites of caldesmon.

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Thøgersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.

Actins↗

PubMed1990

[Electroanalytical chemistry study of pilocarpine].

The electrochemical behaviour of pilocarpine was investigated by differential pulse polarography and cyclic voltammetry. The optimum conditions for the determination of pilocarpine were as follows: The electrolyte was 0.4 mol/L HAc-NaAc(pH 4.0) buffer solution, Zn (II) 1.0 x 10(-3) mol/L; initial potential -0.80 V (vs Ag-AgCl), final potential -1.30 V (vs Ag-AgCl), and voltage sweep rate 5 mV/s. A fair reproducibility in the above procedure and a good relationship between peak current and pilocarpine concentration (4.0 x 10(-6)-4.0 x 10(-5) mol/L) were achieved. The detection limit was 8.0 x 10(-7) mol/L. This paper also reports that the application of cyclic voltammetry to study electrode process behavior has been established. The PVC membrane ion-selective electrode for pilocarpine also was studied. It is based on the use of tetraphenylborate pilocarpine ion pair complex as the active material. The PVC membrane electrode showed Nernstian response over the pilocarpine concentration range from 1.0 x 10(-2) to 3.0 x 10(-5) mol/L, with a slope of 36 mV/decade, the detection limit being 3.0 x 10(-6) mol/L. The electrode gave fast response and good reproducibility. This method provides a rapid and simple way to the determination of pilocarpine in pharmaceutical preparations.

Electrodes↗

PubMed1989

Caldesmon has two calmodulin-binding domains.

Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. C. & Bryan, J., J. Biol. Chem. 261, 16155-16160, 1987; Yazawa, M., Yagi, K. & Sobue, K., J. Biochem. 102, 1065-1073, 1987). In the case of CNBr cleavage a 37 kDa calmodulin-binding fragment (CB40) was obtained. Both CT40 and CB40 contain a reactive thiol group, but these thiols are apparently in different environments as judged by the responses of attached fluorescent labels to calmodulin-binding. A comparison of the N-terminal sequences of CB40 and CT40 with the complete sequence of caldesmon shows that the two calmodulin-binding fragments in fact originate from different parts of the parent molecule. Thus there exist two calmodulin-binding sites in caldesmon, one in the N-terminal half and the other in the C-terminal half of the molecule. This is consistent with the recent finding that up to two calmodulin molecules can be crosslinked to each caldesmon molecule (Wang, C.-L.A., Biochem. Biophys. Res. Commun., 156, 1033-1038, 1988).

Actins↗

PubMed1989

Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1.

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.

Amino Acid Sequence↗

PubMed1988

Towards a biomarker of mammalian senescence: carbonic anhydrase III.

Three proteins (D2, D3, D4) have been identified in the male Fischer 344 rat liver that decrease their concentration dramatically to virtually zero during the transition from physiological maturity to senescence. D3 (Mr 28 kDa), absent (or at a very low concentration) from the livers of newborns and females of all ages, reaches at 60 days (sexual maturity) its maximum concentration, which declines almost linearly thereafter. A homologous protein (CNBr peptide map) occurs in the BALB/c mouse under similar conditions. D3 was purified and since its N-terminal is blocked, digested with CNBr. SDS-PAGE-separated peptides were blotted upon Immobilon and sequenced. The partial sequence matches that of rat carbonic anhydrase III. Treatment of senescent rats with 5 alpha-dihydrotestosterone restores D4 completely, yet D2 and D3 only partially, towards their maximum life-time concentration. Thus senescence-related factors (e.g. hepatic androgen receptor) aside from serum testosterone are responsible for the disappearance of the three proteins from the senescent liver.

Aging↗

PubMed1987

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol.

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.

Animals↗

PubMed1987

The distribution of the charged residues in myosin hinge region and its relationship to the distribution of charged residues in the rest of myosin rod.

Fourier transform analysis of the amino acid sequence of the hinge region from both rabbit skeletal myosin and nematode myosin indicates that the basic residues show strong periodicity of 25.7 residues whereas the periodicity of acidic residues is very weak. Other 100 residue segments of the rod sequence of nematode myosin show an alternation between regions in which the 28 residue repeat is predominantly basic and regions in which it is predominantly acidic. The strong basic and weak acidic near 28 residue repeat of the hinge region appears to form part of this pattern. This alternation suggests that the packing of myosin rods is more favourable when the neighbouring molecules are staggered by about 100 residues or odd multiples of 100 residues, which is consistent with the observed repeats (14.6 and 44.0 nm) of cross-bridges.

Amino Acid Sequence↗

PubMed1986

Changes of lysine reactivities of actin in complex with DNAase I.

The reactivities of lysines of actin in the actin-DNAase I complex were measured by the method of reductive methylation. The reactivities of lysines in the amino-terminal part, lysines 18, 50, 61 and 68, decreased 50%, while those of lysines 237, 283 and 290 increased about 30%, in comparison with those in G-actin, when actin was bound to DNAase I. These results are consistent with the view that the amino-terminal region of actin is the binding site for DNAase I. In conjunction with our earlier work on the reactivities of lysines in F-actin (Lu, R.C. and Szilagyi, L. (1981) Biochemistry 20, 5914-5919), these results are also consistent with the view that DNAase I binds to actin at one of the regions that is involved in polymerization.

Actins↗

PubMed1986

Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides.

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.

Adenosine Diphosphate↗

PubMed1986

Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.

The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.

Adenosine Triphosphatases↗

PubMed1985

Reactivities of thiols in myosin rod: effect of magnesium and ionic strength.

There are six cysteines in each chain of myosin rod of rabbit skeletal muscle: three are in the S-2 portion, at residues 66, 108 and 410 (Lu, R.C. and Lehrer, S. (1984) Biochemistry 23, 5975-5981). The other three are in the light meromyosin portion, assigned at residues 572, 600 and 770 on the basis of homology between the amino acid sequence in the vicinity of these thiols and that of the rod of nematode myosin (McLachlan, A.D. and Karn, J. (1982) Nature 299, 226-231). Since the thiols are distributed in different regions of the rod, measuring their reactivities under various conditions may provide information on the conformations of these regions. Myosin rod was carboxymethylated with radioactive iodoacetic acid under various conditions. The cysteine-containing peptides were isolated using HPLC from the tryptic digests, and the radioactivity incorporated into each thiol was measured. In the denatured state all six thiols were equally reactive. In the native state, all thiols have low reactivity, the reactivity of Cys-108 or -410 is only 0.1% of that in the denatured state, Cys-600 exhibited the highest reactivity, about 20-times that of Cys-410; Cys-66, -572 and -770 had 2-4-times that of Cys-410. When the rods formed filaments, the reactivities of all cysteines further decreased: Cys-66, -108 and -770 were reduced to 50%, while Cys-410, -572 and -600, located in the middle of the rod, were reduced to 20-30% of their reactivities in the monomeric form. In the presence of Mg2+ the reactivity of Cys-108 increased by 20%, whereas Cys-572 decreased by 50%. The results are consistent with the view that metal ions affect the conformation of the rod. This may play a role in the mechanism of filament formation and the movement of crossbridges.

Animals↗

PubMed1985

Reversed-phase high-performance liquid chromatographic separation and quantitation of phenylthiohydantoin derivatives of 25 amino acids, including those of cysteic acid, 4-hydroxyproline, methionine sulfone, S-carboxymethylcysteine and S-methylcysteine.

A high-performance liquid chromatography system is presented which allows separation and quantitation (in the range 4-1000 pmol) of all common phenylthiohydantoin amino acids, including derivatives of 4-hydroxyproline, methionine sulfone and three differently modified forms of cysteine. By showing the actual solvent gradient during elution (as opposed to the programmed gradient) and by supplying information on the effects of minor changes in solvent-pH, column temperature, flow-rate, and concentration of 2-propanol in the gradient, we make guidelines available for fine-tuning the separation with new Ultrasphere-ODS (C18) columns.

Amino Acids↗

PubMed1985

The amino acid sequence and stability predictions of the hinge region in myosin subfragment 2.

From an NH2-terminal sequence analysis of the long and short form of myosin subfragment 2 we have suggested that the putative hinge region in the myosin rod is located in the COOH-terminal portion of the long subfragment 2 (Lu, R. C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2010-2013). The amino acid sequence of this hinge region has now been determined: ASRA KAEKQRSDLSRELEEISERLEEAGGATSAQIEMNK KREAEFEKMRRDLEEATLQHEATAAALRKKHAD SVAELGEQIDNLQRVKQKLEKEKSELKMEIDDLA GNMETVSKAKGNLEKMCRTLEDQ(L/V)SE(V/L)KT KEEEHQRLIN(D/E)L(S/G)AQ(K/R)AR. Comparison of the sequence with that of other portions of the rod, viz. short subfragment 2 and light meromyosin, and of tropomyosin shows that the hinge region shares some feature of a coiled-coil helical structure, but it has somewhat fewer hydrophobic coil-coil interactions and there is a significant number of charged residues in the hydrophobic core region. This suggests that the stability of the putative hinge region would be reduced in comparison with other coiled-coil structures.

Amino Acid Sequence↗

PubMed1984

Effects of interchain disulfide cross-links on the trypsin cleavage pattern and conformation of myosin subfragment 2.

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1984

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.

Amino Acids↗