Biomedical subjects

Y Luo

Publications and source records attributed to Y Luo.

At least 199 records · Page 11Linked to original sources

PubMed1999

The 28-111 disulfide bond constrains the alpha-lactalbumin molten globule and weakens its cooperativity of folding.

Our aim is to determine whether the disulfide bonds of alpha-lactalbumin account for the lack of cooperative folding behavior reported for some molten globule variants, in contrast to the highly cooperative folding reported for the pH 4 molten globule of apomyoglobin. Two different alpha-lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the alpha-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for cooperative folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-111] and [all-Ala]. The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two of the three sets of anion conditions, and anions barely affect its stability. The 28-111 disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Comparison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala]: the unfolding curve of [28-111] is significantly broader. Moreover, the unfolding curves indicate that [28-111] has a lower helix content than [all-Ala]. Consequently, the 28-111 bond constrains the folding behavior of the molten globule and weakens its cooperativity of folding.

Disulfides↗

PubMed1999

Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

Apoptosis↗

PubMed1999

Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation.

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9') and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10-P14, shows facile movement in and out of beta-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the alpha1-antichymotrypsin.alpha-chymotrypsin encounter complex, E.I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of alpha1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E.I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*.

Animals↗

PubMed1999

Collapsin-1/semaphorin-III/D is regulated developmentally in Purkinje cells and collapses pontocerebellar mossy fiber neuronal growth cones.

Most axons in the CNS innervate specific subregions or layers of their target regions and form contacts with specific types of target neurons, but the molecular basis of this process is not well understood. To determine whether collapsin-1/semaphorin-III/D, a molecule known to repel specific axons, might guide afferent axons within their cerebellar targets, we characterized its expression by in situ hybridization and observed its effects on mossy and climbing fiber extension and growth cone size in vitro. In newborn mice sema-D is expressed by cerebellar Purkinje cells in parasagittal bands located medially and in some cells of the cerebellar nuclei. Later, sema-D expression in Purkinje cells broadens such that banded expression is no longer prominent, and expression is detected in progressively more lateral regions. By postnatal day 16, expression is observed throughout the cerebellar mediolateral axis. Collapsin-1 protein, the chick ortholog of sema-D, did not inhibit the extension of neurites from explants of inferior olivary nuclei, the source of climbing fibers that innervate Purkinje cells. In contrast, when it was applied to axons extending from basilar pontine explants, a source of mossy fiber afferents of granule cells, collapsin-1 caused most pontine growth cones to collapse, as evidenced by a reduction in growth cone size of up to 59%. Moreover, 63% of pontine growth cones arrested their extension or retracted. Its effects on mossy fiber extension and its distribution suggest that sema-D prevents mossy fibers from innervating inappropriate cerebellar target regions and cell types.

Animals↗

PubMed1999

Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB.

The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

Apoptosis↗

PubMed1999

Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.

It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+ dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+ in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC.TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.

Actins↗

PubMed1999

Fibroblast growth factor-10. A second candidate stromal to epithelial cell andromedin in prostate.

Fibroblast growth factor (FGF)-10, a homologue of FGF-7, is expressed significantly in normal rat prostate tissue, well differentiated rat prostate tumors with an epithelial and stromal compartment and only in derived prostate stromal cells in culture. Similar to FGF-7, recombinant rat FGF-10 was a specific mitogen for prostate epithelial cells. In contrast to FGF-7 which is widely expressed among stromal cells in tissues, the expression of FGF-10 correlated with the presence of stromal cells of muscle origin. Radioreceptor binding assays and covalent cross-linking analysis revealed that FGF-10 binds with an affinity equal to FGF-7 to resident epithelial cell receptor, FGFR2IIIb, but unlike FGF-7 also binds the IIIb splice variant of FGFR1. Analysis of mRNA expression by RNase protection revealed that, similar to FGF-7, the expression of FGF-10 was responsive to androgen in stromal cells from normal prostate and non-malignant differentiated tumors. Although FGF-10 cDNA exhibits a signal sequence for secretion, cultured stromal cells exhibit strictly a cell-associated FGF-10 antigen that correlates with an alternately translated intracellular isoform. FGF-10 requires 1.4 times higher NaCl for elution from immobilized heparin than does FGF-7 and binds to four times the number of sites on the pericellular matrix of epithelial cells. The results show that prostate stromal cell-derived FGF-10, like FGF-7, exhibits the properties of an andromedin which may indirectly mediate control of epithelial cell growth and function by androgen. Although FGF-10 and FGF-7 bind and activate the same resident epithelial cell receptor (FGFR2IIIb), differences in cell type of origin, compartmentation by alternate translation, the affinity for FGFR1IIIb, and access to FGFR by differential interaction with pericellular matrix heparan sulfate suggest they may play both independent and compensatory roles in prostate homeostasis.

Animals↗

PubMed1999

Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor.

The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.

Animals↗

PubMed1999

Discovery of a regulatory motif that controls the exposure of specific upstream cyclin-dependent kinase sites that determine both conformation and growth suppressing activity of pRb.

The conformation and activity of pRb, the product of the retinoblastoma susceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is not clear whether the phosphorylation status of one or more of these sites contributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the phosphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusual structural motif, m89 (amino acids 880-900), the mutation of which confers upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function of pRb. In order to understand the effect of the mutation of m89 on the phosphorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in combinations results in the different conformations of pRb, the migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of these hypophosphorylated forms of pRb has enhanced growth suppressing activity relative to the wild type. Our data revealed that the m89 structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant has enhanced growth suppressing activity, similar to a mutant with alanine substitutions at Ser-807/Ser-811. Our recent finding, that the m89 region is part of a structural domain, p5, conserved antigenically and functionally between pRb and p53, suggests that the evolutionarily conserved p5 domain may play a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.

Amino Acid Sequence↗

PubMed1999

[Study on the relationship between the structure of chlorobenzenes and their toxicity to guppy].

The quantum chemical indexes of 12 chlorobenzenes were calculated by CNDO/2 method. Quantitative structure-activity relationship between the quantum chemical indexes of chlorobenzenes and their toxicity to Guppy was calculated by step regression analysis. The linear correlation equation is as follows: DLC50 = -0.15 + 6.865 sigma QR, n = 12, R = 0.9867 s = 0.15, F = 367.64. Results showed that the toxicity of chlorobenzenes to the aquatic Guppy are increased as the increase of their ability to accept electrons.

Animals↗

PubMed1999

Sequence analysis of Pasteurella multocida major outer membrane protein (OmpH) and application of synthetic peptides in vaccination of chickens against homologous strain challenge.

Pasteurella multocida major outer membrane protein (OmpH) has been previously characterized as a porin. The native OmpH from strain X-73 (serotype 1) but not recombinant protein from Escherichia coli induced homologous protection in chickens. In this study OmpH sequences from 15 P. multocida serotypes as well as the CU vaccine strain were compared by sequence alignment and revealed high homology, with major variations confined to two discrete regions which were correspondingly predicted as two largest external loops. Secondary structures of OmpHs were predicted by sequence alignment of OmpHs with well defined porins and analyses of amphiphilicity, hydrophobic moment and antigenic index plots. Several synthetic peptides derived from predicted loop 2 and loop 5 of X-73 OmpH were synthesized as vaccine candidates. Vaccination studies in chickens showed that the cyclic synthetic peptide (Cyclic-L2) mimicking the predicted loop 2 induced 70% protection in chickens against strain X-73 challenge. This is the first report that a synthetic peptide mimicking the conformational epitopes of a native protein provide practical protection in target animal against bacterial infection.

Amino Acid Sequence↗

PubMed1999

IFN-alpha 2B enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette-Guérin immunotherapy.

Combination therapy with intravesical bacillus Calmette-Guérin (BCG) plus IFN-alpha for superficial bladder cancer has been demonstrated to be more effective than either single agent alone in animal studies and of suggested greater efficacy in clinical studies. However, the mechanism by which IFN-alpha enhances BCG-mediated antitumor activity is poorly understood. Using PBMCs from bladder cancer patients, IFN-alpha was found to substantially enhance the efficacy of BCG to induce IFN-gamma production. Among 34 patients tested, 80% showed >4-fold increase. This effect of IFN-alpha was observed in both initial and memory responses to BCG. In addition, IFN-alpha up-regulated BCG-induced IL-12 and TNF-alpha and down-regulated BCG-induced IL-10. Neutralizing endogenous IL-10 or adding exogenous IL-12 provided further synergy for IFN-gamma production. In clinical practice, intravesical IFN-alpha 2B (50 million units (MU)/dose) was observed to accelerate urinary IFN-gamma production to low-dose BCG (one-tenth or one-third of a full dose) in patients treated with combination therapy compared with BCG alone. These results suggest that IFN-alpha is a potent BCG enhancer that polarizes the BCG-induced immune response toward the cellular immune pathway by promoting Th1 cytokine expression and reducing Th2 cytokine expression. This study provides an immunological basis for future rational use of IFN-alpha in conjunction with intravesical BCG for bladder cancer immunotherapy.

Adjuvants, Immunologic↗

PubMed1999

The 1.8 A structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism.

Intramolecular trans-sialidase from leech (Macrobdella decora) is the first member of the sialidase superfamily found to exhibit strict specificity towards the cleavage of terminal Neu5Acalpha2-->3Gal linkage in sialoglycoconjugates. Its release of 2,7-anhydro-Neu5Ac instead of Neu5Ac indicates that it catalyzes an intramolecular trans-sialosyl reaction. Crystal structures of its complexes with an inactive substrate analogue 2-propenyl-Neu5Ac, and with the product 2,7-anhydro-Neu5Ac, have been determined to 1.8 A resolution. The boat conformation of the pyranose observed in the complexes supports the proposed enzymatic mechanism that O7 of an axial 6-glycerol group attacks the positively charged C2 of the intermediate. A generalized mechanism is proposed for the sialidase superfamily.

Amino Acid Sequence↗

PubMed1999

Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications.

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.

Base Sequence↗

PubMed1999

Upregulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipoprotein receptor function.

This study examines the effect of mutation of the low-density lipoprotein receptor (LDLR) on cholesterol metabolism, and especially lipoprotein-derived cholesteryl ester uptake, in murine ovarian granulosa cells. Although the tests were conducted on cells prepared by two different procedures, the results are similar. Deletion of LDLR function did not noticeably affect key enzymes of the steroidogenic pathway or affect progestin production and secretion in granulosa cells. No change was found in expression of LDL-related protein (LRP). These data suggested that cholesterol turnover in cells from the knockout animals is within normal limits and that the cells are not stressed to acquire more cholesterol. Both biochemical and morphological data indicate that unstimulated granulosa cells from LDLR-/- mice are nonetheless programmed to take in double the amount of lipoprotein-derived cholesteryl ester (via the selective cholesteryl ester uptake pathway) and to process (hydrolyze, re-esterify, or utilize) more than twofold the cholesteryl ester processed by cells from wildtype (LDLR+/+) animals. Bt2cAMP stimulation of the murine granulosa cells increases the mass of cholesteryl ester taken up by the selective pathway by an additional 38%. To determine to what extent this increase is related to high-density lipoprotein (HDL) scavenger receptor protein (SR-BI) or caveolin function, Western blots and immunohistochemical studies were performed under a variety of conditions. SR-BI levels are found to be low in unstimulated cells of both LDLR+/+ and LDLR-/- animals, but highly expressed (approximately 20-fold increase over basal levels) in stimulated (Bt2cAMP) cells of both animal models. Thus, the functional relationship between selective cholesteryl ester uptake and SR-BI receptor protein is not as tight as in previously reported studies, suggesting a requirement for other tissue factors. Caveolin expression did not change under any of the conditions tested and appears not to be functionally involved in this process.

Animals↗

PubMed1999

Interleukin-2 receptoralpha gene expression in intestinal allograft rejection.

This study was undertaken to determine whether the interleukin-2 receptor alpha (IL-2Ralpha) coding gene could serve as a genetic marker of intestinal allograft rejection. The small bowel graft from Jinbai mice was heterotopically transplanted to BALB/c mice. Naive BALB/c mice and BALB/c mice with intestinal isografts were used as controls. The mRNA transcription level of the spleen cells harvested from the recipients and controls was measured with quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) on postoperative day (POD) 3 and routine pathology was studied in these grafts. The IL-2Ralpha mRNA transcription level in untreated allografts was significantly higher than that of controls on POD 3, when intestinal allograft rejection was not detected. These data suggest that the IL-2Ralpha mRNA transcription level may reflect the early cellular immune response and this genetic marker may be of value in monitoring intestinal allograft rejection.

Animals↗

PubMed1999

In vivo observation of lactate methyl proton magnetization transfer in rat C6 glioma.

Magnetic resonance spectroscopy (MRS) measurements of the lactate methyl proton in rat brain C6 glioma tissue acquired in the presence of an off-resonance irradiation field, analyzed using coupled Bloch equation formalism assuming two spin pools, demonstrated the occurrence of magnetization transfer. Quantitative analysis revealed that a very small fraction of lactate (f = 0.0012) is rotationally immobilized despite a large magnetization transfer effect. Off-resonance rotating frame spin-lattice relaxation studies demonstrated that deuterated lactate binds to bovine serum albumin and the proteins present in human plasma, thereby providing a possible physical basis for the observed magnetization transfer effect. These results demonstrate that partial or complete saturation of the motionally restricted lactate pool (as well as other metabolites) by the application of an off-resonance irradiation field, such as that used for water presaturation, can lead to a substantial decrease in resonance intensity by way of magnetization transfer effects, resulting in quantitation errors.

Animals↗

PubMed1999

Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.

Adenosine Triphosphate↗