Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process.

Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.

Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling.

Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24-48 h in a medium containing insulin (5 microg/ml) and leptin (3, 10, and 62 nm) or IL-1beta (0.6, 3, and 10 nm) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1beta also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin's role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).

Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures.

Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

Regulation of protein kinase C by the cytoskeletal protein calponin.

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide.

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCalpha and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain-an essential region for caveolin interaction with signaling molecules--significantly inhibited agonist-induced translocation of both PKCalpha and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCalpha and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.

Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actin-binding proteins.

An interaction between extracellular regulated kinase 1 (ERK1) and calponin has previously been reported (Menice, Hulvershorn, Adam, Wang and Morgan (1997) J. Biol. Chem. 272 (40), 25157-25161) and has been suggested to reflect a function of calponin as a signalling molecule. We report in this study that calponin binds to both ERK1 and ERK2 under native conditions as well as in an overlay assay. Using chymotryptic fragments of calponin, the binding site of ERK on calponin was identified as the calponin homology (CH) domain, an N-terminal region of calponin found in other actin-binding proteins. ERK also bound, in a gel overlay assay, alpha-actinin, a protein with two tandem CH domains, as well as a 27 kDa thermolysin product of alpha-actinin containing the CH domains of alpha-actinin. The CH domain of calponin could compete with intact calponin or alpha-actinin for ERK binding. Titration of acrylodan-labelled calponin with ERK gave a K(a) of 6x10(6) M(-1) and titration of acrylodan-labelled calponin with a peptide from the alphaL16 helix of ERK gave a K(a) of 1x10(6) M(-1). Recombinant ERK was found to co-sediment with purified actin and induced a fluorescence change in pyrene-labelled F-actin (K(a)=5x10(6) M(-1)). The interaction of ERK with CH domains points to a new potential function for CH domains. The interaction of ERK with actin raises the possibility that actin may provide a scaffold for ERK signalling complexes in both muscle and non-muscle cells.

Progress in characterization of pre-implantation factor in embryo cultures and in vivo.

PROBLEM: Pre-implantation factor (PIF), a small, embryo-derived peptide is detected in the maternal serum prior to implantation and is associated with successful pregnancy outcome. However, its identity is not known. METHOD OF STUDY: PIF was isolated from mouse embryo conditioned media and from pregnant porcine sera, using high-performance liquid chromatography (HPLC) followed by mass spectrometry. RESULTS: Conditioned culture media was separated by gel filtration chromatography followed by reversed phase chromatography. At each step, PIF activity was determined by the lymphocyte/platelet binding autorosette assay (LPBA). Mass spectrometry yielded a single peak with a mass of 1300 Da. The peptide is, however, present in very low concentrations (fM), which has so far precluded complete identification. Pregnant porcine sera that exhibit potent PIF activity were deproteinated by acetone and further fractionated by reversed phase HPLC. Active fractions contain peptides of molecular masses 523 and 551 Da. CONCLUSION: PIF, likely to be peptides, represents a novel substance related to pregnancy initiation and maintenance.

Interaction of deletion mutants of troponins I and T: COOH-terminal truncation of troponin T abolishes troponin I binding and reduces Ca2+ sensitivity of the reconstituted regulatory system.

The interaction between troponin I (TnI) and troponin T (TnT) remains the least understood binary interaction among the regulatory proteins of vertebrate striated muscle. To identify the specific binding domains of TnI and TnT and to evaluate the interactions of TnT with troponin C and tropomyosin (Tm), we generated an NH2-terminal fragment of human fast skeletal beta TnT (TnT1-201; residues 1-201) using site-directed mutagenesis. The mutant protein failed to bind to rabbit skeletal muscle TnI as judged by HPLC, showed reduced TnC binding and reduced ternary troponin (Tn) complex formation, and exhibited a much reduced Ca2+ sensitivity in the reconstituted regulatory system. It is shown that the amount of Tn complex formed by TnT1-201 rather than the activity of the mutant Tn complex affected this Ca2+ sensitivity. Binding of the mutant to Tm was similar to that of intact TnT. These results support the view that the COOH-terminal segment of TnT is necessary for binding to TnI and TnC and Ca2+ sensitivity in the thin filament, whereas its NH2-terminus strongly binds to Tm. To identify the regions of TnI which bind to muscle TnT, we used four recombinant fragments of fast skeletal muscle TnI containing amino acid residues 1-94 (TnI1-94), 1-120 (TnI1-120), 96-181 (TnI96-181), and 122-181 (TnI122-181) and a synthetic peptide, TnI98-114, containing residues 98-114 corresponding to the inhibitory region. Only TnI1-120 showed weak binding to TnT but not to TnT1-201. These results suggest that (i) a region within the NH2-terminal 120 residues of TnI interacts with TnT and (ii) the COOH-terminal residues 202-258 of TnT contain the interaction site of TnI. Overall, our results also imply that residues 159-201 constitute the smallest region of TnT which contributes to the Ca2+ sensitivity of actoS1 ATPase in a reconstituted regulatory system.

Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization.

Preimplantation factor (PIF) is detected in the serum of women shortly after fertilization; its origin, however, has not been established. In this study, the embryonal origin of PIF was investigated and partial characterization of the factor was carried out. Culture media from viable human 2-8-cell stage embryos and mouse 2-cell-blastocyst stage embryos were analysed using the lymphocyte/platelet binding assay (LPBA). The assay was performed by combining culture media with donor O+ type blood-derived lymphocytes/platelets, complement and an antibody against CD2. Increased autorosette formation between lymphocytes and platelets (> 9%) was an indication for the presence of PIF. In addition, the effect of platelet-activating factor (PAF) and chaperonin 10 on PIF activity was determined. Partial purification of PIF was carried out using gel filtration and reverse-phase high purification liquid chromatography (HPLC), followed by mass spectrometry. Culture media of single human viable fertilized oocytes were negative for PIF; however, the 10-fold concentrated medium was positive for PIF. In medium in which five or more mouse embryos were cultured, PIF activity was observed starting at the morula stage and was higher by the blastocyst stage. Addition of PAF or chaperonin 10 to the PIF assay did not elicit a specific effect on PIF activity. Chromatographic data suggest that PIF activity is due to low molecular weight proteins. PIF appears to be a low molecular weight protein which is derived from viable preimplantation embryos. It is different from PAF or chaperonin 10. Its final characterization will be valuable for better understanding of maternal recognition of pregnancy and implantation.

In vitro activation of a 60-70 kDa histone H4 protein kinase from neutrophils by limited proteolysis.

Neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP) are known to exhibit a rapid and transient activation of a histone H4 kinase that may function in a stimulatory pathway downstream of phosphatidylinositol 3-kinase. The activity of this histone kinase in unstimulated neutrophils and cells treated with 1.0 microM fMLP for 10 sec was 8.8 +/- 5 and 43 +/- 2 pmol P/min per 10(7) cells, respectively. In this paper, we report that unstimulated neutrophils contain a latent H4 kinase in the 100,000 x g soluble fraction that can be markedly activated by treatment with trypsin. The values for the untreated and trypsin treated enzyme were 5.5 +/- 1.0 and 63.6 +/- 18 pmol P/min per 10(7) cell-equivalents, respectively. This kinase was insensitive to a selective antagonist of protein kinase C (i.e., 50 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) but completely blocked by 100 nM staurosporine. Only a single peak of activity was observed for this enzyme when the 100,000 x g supernatant fraction was fractionated on either an exclusion (KW-803) or an anion exchange column (DEAE), or during isoelectric focusing. The molecular weight of the latent kinase was 64 +/- 6 kDa and the isoelectric point was 7.6 +/- 0.1. During all fractionation procedures, the H4 kinase co-chromatographed with a trypsin-activated kinase that catalyzed the phosphorylation of a peptide which corresponds to residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox). The properties of the trypsin-activated H4 kinase from unstimulated neutrophils are very similar to those reported for this enzyme from fMLP-stimulated cells.

Interaction of a troponin I inhibitory peptide with both domains of troponin C.

Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.

Overproduction and rapid purification of human fast skeletal beta troponin T using Escherichia coli expression vectors: functional differences between the alpha and beta isoforms.

Troponin T (TpnT), an essential component of the Ca(2+)-regulatory troponin complex, is involved in protein-protein interactions with other thin-filament proteins during muscle contraction in vertebrate striated muscle (VSM). The isoforms of TpnT are encoded by members of a multigene family which, by alternate splicing, produces a complex pattern of isoproteins in VSM. The functional domains of TpnT are only tentatively identified and structure-function analysis on this protein is limited due to the heterogeneity of the multiple isoforms. We reasoned that the overproduction and purification of a single TpnT species in Escherichia coli would provide an insight into these studies, besides being useful in crystallizing the protein. We cloned the human fast skeletal beta TpnT-encoding cDNA (beta TpnTf) in three expression vectors. Overexpression was achieved in an E. coli BL21 (DE3) lysogen using a T7 RNA polymerase promoter-based vector, pET17b. The unfused recombinant protein was purified by a simple and rapid procedure in a biologically active and immunoreactive form. This is the first successful synthesis of a complete beta TpnTf polypeptide from any species using an in vitro expression system. Purified human beta TpnTf, a predominant fetal form, was less Ca(2+)-sensitive and exhibited considerably reduced affinity for troponin C and tropomyosin, as compared to the rabbit fast skeletal alpha TpnT, a predominant adult isoform. These results provide a biochemical correlate to the age-related differences in Ca2+ sensitivity of tension development in vertebrate fast skeletal muscles.

Overexpression and rapid purification of rabbit fast skeletal troponin I from Escherichia coli: effect of different promoters, host strains, and culture conditions.

Rabbit fast skeletal troponin I (TnIf) cDNA was expressed using two Escherichia coli expression vectors, pRE1 containing the bacteriophage lambda pL promoter and pAED4, a T7 RNA polymerase-based vector. Although both vectors expressed TnIf, overexpression of the target protein was achieved with pAED4. The effect of several parameters such as culture condition, compatible host strain, and inhibition of protein synthesis by rifampicin on the expression of TnIf was investigated. The overexpressed target protein synthesized during a brief induction period of only 2 h was conveniently purified from inclusion bodies by a simple and rapid procedure involving extraction with urea, ultracentrifugation, DE-52 column chromatography, and gel filtration. About 50-75 mg of highly purified TnIf was obtained per liter E. coli culture by this method, which does not involve time-consuming multistep procedures such as affinity and ion exchange chromatography as previously reported in the literature. The isolated unfused protein is stable and is indistinguishable from native protein in all biological parameters examined. The parameters optimized in this report for overexpression of TnIf may also be applicable for other eukaryotic proteins.

A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family.

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.

Characterization of a rabbit fast skeletal troponin I cDNA: a comparative sequence analysis of vertebrate isoforms and tissue-specific expression of a single copy gene.

A cDNA encoding skeletal fast troponin I (TnIf) has been isolated and characterized from an adult rabbit fast skeletal muscle cDNA library. This cDNA contains the entire coding region of 549 base pairs (bp), the 3' untranslated (UT) segment of 78 bp and the 5' UT segment of 74 bp. The 3' UT segment shows the predicted stem-loop structure characteristic of eukaryotic mRNAs. The cDNA-derived amino acid sequence is not identical to the chemically-derived and the recently reported cDNA-derived amino acid sequences for rabbit TnIf, and thus enables to make the necessary corrections in the sequence information. The rabbit TnIf is encoded by a single copy gene which is expressed in adult fast skeletal muscle in a tissue-specific manner. Comparative sequence analysis of the vertebrate TnI isoforms and their cDNAs shows a high level of sequence conservation in the coding region among the TnIf interspecies isoforms. The sequence similarity is markedly decreased among the fast, slow and cardiac intra- and inter-species isoforms. The C-terminal halves of TnI isoforms including a segment believed to be involved in critical interactions with actin and troponin C (TnC) show high sequence conservation whereas the N-terminal halves show considerable difference in sequence and size. The significance of these results in relation to the biological function and evolution of members of the vertebrate TnI multigene family is discussed.

Quantitation of liquid-crystalline ordering in F-actin solutions.

Actin filaments (F-actin) are important determinants of cellular shape and motility. These functions depend on the collective organization of numerous filaments with respect to both position and orientation in the cytoplasm. Much of the orientational organization arises spontaneously through liquid crystal formation in concentrated F-actin solutions. In studying this phenomenon, we found that solutions of purified F-actin undergo a continuous phase transition, from the isotropic state to a liquid crystalline state, when either the mean filament length or the actin concentration is increased above its respective threshold value. The phase diagram representing the threshold filament lengths and concentrations at which the phase transition occurs is consistent with that predicted by Flory's theory on solutions of noninteracting, rigid cylinders (Flory, 1956b). However, in contrast to other predictions based on this model, we found no evidence for the coexistence of isotropic and anisotropic phases. Furthermore, the phase transition proved to be temperature dependent, which suggests the existence of orientation-dependent interfilament interactions or of a temperature-dependent filament flexibility. We developed a simple method for growing undistorted fluorescent acrylodan-labeled F-actin liquid crystals; and we derived a simple theoretical treatment by which polarization-of-fluorescence measurements could be used to quantitate, for the first time, the degree of spontaneous filament ordering (nematic order parameter) in these F-actin liquid crystals. This order parameter was found to increase monotonically with both filament length and concentration. Actin liquid crystals can readily become distorted by a process known as "texturing." Zigzaging and helicoidal liquid crystalline textures which persisted in the absence of ATP were observed through the polarizing microscope. Possible texturing mechanisms are discussed.

Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

Distance measurements in cardiac troponin C.

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.