Laboratory longitudinal diffusion tests: 2. Parameter estimation by inverse analysis.

This study focuses on the verification of test interpretations for different state analyses of diffusion experiments. Part 1 of this study identified that steady, quasi-steady and equilibrium state analyses for the through- and in-diffusion tests with solution reservoirs are generally feasible where the tracer is not highly sorptive. In Part 2 we investigate parameter identifiability in transient-state analysis of reservoir concentration variation using a numerical approach. For increased generality, the analytical models, objective functions and Jacobian matrix necessary for inverse analysis of transient-state data are reformulated using unified dimensionless parameters. In these dimensionless forms, the number of unknown parameters is reduced and a single dimensionless parameter represents the sorption property. The dimensionless objective functions are evaluated for individual test methods and parameter identifiability is discussed in relation to the sorption property. The effects of multiple minima and measurement error on parameter identifiability are also investigated. The main findings are that inverse problems for inlet and outlet reservoir concentration analyses are generally unstable and well-posed, respectively. Where the tracer is sorptive, the inverse problem for the inlet reservoir concentration analysis may have multiple minima. When insufficient measurement data is collected, multiple solutions may result and this should be taken into consideration when inversely analyzing data including that of inlet reservoir concentration. Verification of test interpretation by cross-checking different state analyses is feasible where the tracer is not highly sorptive. In an actual experiment, test interpretation validity is demonstrated through consistency between theory and practice for different state analyses.

Laboratory longitudinal diffusion tests: 1. Dimensionless formulations and validity of simplified solutions.

To obtain reliable diffusion parameters for diffusion testing, multiple experiments should not only be cross-checked but the internal consistency of each experiment should also be verified. In the through- and in-diffusion tests with solution reservoirs, test interpretation of different phases often makes use of simplified analytical solutions. This study explores the feasibility of steady, quasi-steady, equilibrium and transient-state analyses using simplified analytical solutions with respect to (i) valid conditions for each analytical solution, (ii) potential error, and (iii) experimental time. For increased generality, a series of numerical analyses are performed using unified dimensionless parameters and the results are all related to dimensionless reservoir volume (DRV) which includes only the sorptive parameter as an unknown. This means the above factors can be investigated on the basis of the sorption properties of the testing material and/or tracer. The main findings are that steady, quasi-steady and equilibrium-state analyses are applicable when the tracer is not highly sorptive. However, quasi-steady and equilibrium-state analyses become inefficient or impractical compared to steady state analysis when the tracer is non-sorbing and material porosity is significantly low. Systematic and comprehensive reformulation of analytical models enables the comparison of experimental times between different test methods. The applicability and potential error of each test interpretation can also be studied. These can be applied in designing, performing, and interpreting diffusion experiments by deducing DRV from the available information for the target material and tracer, combined with the results of this study.

Hotspots of MLV integration in the hematopoietic tumor genome.

Extensive research has been performed regarding the integration sites of murine leukemia retrovirus (MLV) for the identification of proto-oncogenes. To date, the overlap of mutations within specific oligonucleotides across different tumor genomes has been regarded as a rare event; however, a recent study of MLV integration into the oncogene Zfp521 suggested the existence of a hotspot oligonucleotide for MLV integration. In the current review, we discuss the hotspots of MLV integration into several genes: c-Myc, Stat5a and N-myc, as well as ZFP521, as examined in tumor genomes. From this, MLV integration convergence within specific oligonucleotides is not necessarily a rare event. This short review aims to promote re-consideration of MLV integration within the tumor genome, which involves both well-known and potentially newly identified and novel mechanisms and specifications.

Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

ZFP521 contributes to pre-B-cell lymphomagenesis through modulation of the pre-B-cell receptor signaling pathway.

ZFP521 was previously identified as a putative gene involved in induction of B-cell lymphomagenesis. However, the contribution of ZFP521 to lymphomagenesis has not been confirmed. In this study, we sought to elucidate the role of ZFP521 in B-cell lymphomagenesis. To this end, we used a retroviral insertion method to show that ZFP521 was a target of mutagenesis in pre-B-lymphoblastic lymphoma cells. The pre-B-cell receptor (pre-BCR) signaling molecules BLNK, BTK and BANK1 were positively regulated by the ZFP521 gene, leading to enhancement of the pre-BCR signaling pathway. In addition, c-myc and c-jun were upregulated following activation of ZFP521. Stimulation of pre-BCR signaling using anti-Vpreb antibodies caused aberrant upregulation of c-myc and c-jun and of Ccnd3, which encodes cyclin D3, thereby inducing the growth of pre-B cells. Stimulation with Vpreb affected the growth of pre-B cells, and addition of interleukin (IL)-7 receptor exerted competitive effects on pre-B-cell growth. Knockdown of BTK and BANK1, targets of ZFP521, suppressed the effects of Vpreb stimulation on cell growth. Furthermore, in human lymphoblastic lymphoma, analogous to pre-B-cell lymphoma in mice, the expression of ZNF521, the homolog of ZFP521 in humans, was upregulated. In conclusion, our data showed that the ZFP521 gene comprehensively induced pre-B-cell lymphomagenesis by modulating the pre-B-cell receptor signaling pathway.

A simple method for the separation of cardiac myosin light chains.

A simple and efficient procedure is presented for the separation of pig cardiac myosin light chains. The method employs only three repetitive isoelectric precipitations without column chromatography, and is suitable for use with large quantities of material. The method also permits the recovery of homogeneous light chains with good yields.

New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.

The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives.

The ribose-modified chromophoric and fluorescent analog of ATP 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293-297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.

Fluorescence properties of 2' (or 3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate and its use in the study of binding to heavy meromyosin ATPase.

2' (or 3')-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (N3ph-ATP), which contains a Meisenheimer complex moiety, is one of the class of compounds which do not fluoresce in water but fluoresce both in low polarity solvents and when bound to the protein molecule. Fluorescence intensity of N3ph-ATP in the range of 540 nm, when excited at 410 nm, decreased with increasing the solvent polarity accompanying the increment of the wavelength of maximum emission. When bound to heavy meromyosin ATPase, the fluorescence properties of N3ph-ADP were almost the same as those of N3ph-ATP in a low polarity solvent, suggesting that N3ph-ADP was bound to hydrophobic area on heavy meromyosin ATPase.

Preventive therapy for non-steroidal anti-inflammatory drug-induced ulcers in Japanese patients with rheumatoid arthritis: the current situation and a prospective controlled-study of the preventive effects of lansoprazole or famotidine.

BACKGROUND: There is a lack of evidence for the efficacy of preventive medications for peptic ulcers (PUs) among long-term users of non-steroidal anti-inflammatory drugs (NSAIDs) in Japan. AIM: To estimate the preventive effect by normal dose, not high-dose histamine-H2 receptor antagonists (H2RA) for NSAID-induced ulcers. METHODS: We designed two different studies to assess the efficacy of anti-ulcer agents in rheumatoid arthritis (RA) in patients treated over a long term with NSAIDs. An investigative survey divided patients into those not taking anti-ulcer agents (non-medication group); those taking mucosal protective agents (mucosal protectant group), H2RA (H2RA group), proton pump inhibitors (PPI group), or a prostaglandin E1 analog (PG) (PG group). The second study compared prospectively the preventive effects of either famotidine 20 mg bd (famotidine group) or lansoprazole 15 mg daily (lansoprazole group) in patients with PU scars. RESULTS: The prevalence of PU in the H2RA group was significantly lower compared to the mucosal protectant group (P < 0.05), and the mucosal protectant group was not significantly different to the non-medication group. The prospective study revealed that the PU onset rate of the famotidine group was 8% (1/13), and lansoprazole group was 15% (2/13), indicating no significant differences between the two. CONCLUSIONS: In Japan, normal-dose H2RA is expected to be a new PU preventive treatment strategy in patients requiring long-term NSAID therapy.

Implications of corpus gastritis, atrophy and cyclooxygenase in the development of gastric erosions after curing Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori eradication decreases recurrence of peptic ulcers with marked improvement in histological inflammation, but gastric mucosal injuries may be developed even after eradication. PURPOSE: To investigate the mechanisms responsible for the development of gastric erosions after eradication, we analysed the relationship between clinicopathological risk factors and the occurrence of gastric erosion after curing H. pylori infection. PATIENTS: Sixty patients underwent endoscopy before, and 3, 6 and 12 months after the completion of H. pylori eradication. METHODS: Risk factors associated with the development of gastric erosions after eradication were assessed by multivariate analysis, and cyclooxygenase-1 and -2 immunoreactivity was histologically examined in the gastric mucosa before and after eradication. RESULTS: The cumulative prevalence of gastric erosions after H. pylori eradication was 38.3% within 1 year. Using multivariate analysis, corpus gastritis scores (inflammation score+activity score), corpus atrophy scores and an age of more than 50 years were found to be independent factors associated with the development of gastric erosion after eradication with odds ratios of 7.39, 0.13 and 5.00, respectively. Cyclooxygenase-2 immunoreactivity of the corpus was decreased for the non-erosion group after eradication, but not for the erosion group. CONCLUSIONS: Severe gastritis or less severe atrophy in oxyntic glands but not in pyloric glands before eradication may be involved in the development of gastric erosions after curing H. pylori infection.

Inhibition of Helicobacter pylori-induced cyclo-oxygenase-2 aggravates NSAID-caused gastric damage in Mongolian gerbils.

BACKGROUND: The effect of Helicobacter pylori infection on non-steroidal anti-inflammatory drug-induced gastric mucosal injury is controversial. AIM: To examine the effect of the interaction between H. pylori and non-steroidal anti-inflammatory drugs on gastric mucosal injury. METHODS: Mongolian gerbils infected with H. pylori were treated with indometacin at 8 mg/kg for 2 days or 7 days. Mucosal damage was assessed by macroscopic and histological examination, and myeloperoxidase activity was measured as an index of neutrophil infiltration. The expression levels of cyclo-oxygenase proteins were determined by Western blot analysis and cyclo-oxygenase activity. RESULTS: A 2-day course of indometacin did not cause an increase in gastric damage in H. pylori-infected Mongolian gerbils compared to uninfected gerbils, while a 7-day course of indometacin caused additive gastric damage in H. pylori-infected animals. H. pylori infection induced cyclo-oxygenase-2 expression in the stomach. Treatment with indometacin for 2 days did not significantly affect cyclo-oxygenase activity in H. pylori-infected animals, while treatment for 7 days inhibited both cyclo-oxygenase-1 and cyclo-oxygenase-2 activities. Pre-treatment with a selective cyclo-oxygenase-2 inhibitor aggravated mucosal injury in H. pylori-infected animals treated or not treated with indometacin for 2 days. CONCLUSIONS: Our results suggest that cyclo-oxygenase-2 protein induced by H. pylori infection may be involved in the defence of the gastric mucosa against damage caused by non-steroidal anti-inflammatory drugs. Therefore, inhibition of cyclo-oxygenase-2 activity may enhance non-steroidal anti-inflammatory drug-caused gastric damage in H. pylori-infected animals.

Effect of acid suppression therapy on development of gastric erosions after cure of Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori eradication markedly improves histological inflammation and decreases peptic ulcer recurrence, but little is known about the subsequent development of gastric mucosal injury. AIM: To investigate whether acid suppression treatment after eradication influences the development of gastric erosions. METHODS: Eighty-one patients (gastritis or peptic ulcer) after successful H. pylori eradication were divided into two groups: 40 received an H2-blocker for 6 months (H2-blocker-positive) and 41 received no treatment (H2-blocker-negative). Endoscopy was performed before, and at 3 and 6 months after completion of eradication. RESULTS: Cumulative prevalence of gastric erosions in the H2-blocker-positive group was significantly lower than in the H2-blocker-negative group, 25% vs. 42%, respectively. In the H2-blocker-negative group but not the H2-blocker-positive group, the cumulative prevalence of gastric erosions after eradication was higher in patients with less severe corpus atrophy or more severe corpus gastritis. CONCLUSIONS: Development of gastric erosions after H. pylori eradication may be controlled by acid suppression treatment. Less severe atrophy or more severe gastritis in oxyntic glands before eradication may be involved in the development of gastric erosions. These results support the idea that recovery of acid secretion may be one of factors for development of gastric mucosal erosions after successful eradication.

Plasma and BAL fluid concentrations of antimicrobial peptides in patients with Mycobacterium avium-intracellulare infection.

STUDY OBJECTIVES: To investigate the roles of human alpha-defensin (HAD), human beta-defensin (HBD)-1, and HBD-2, novel antimicrobial peptides, in patients with Mycobacterium avium-intracellulare infection (MAI). PATIENTS: The study included 25 patients (10 men) with MAI who visited our hospital between June 1998 and August 1999. MEASUREMENTS AND RESULTS: In patients with pulmonary MAI, we measured HAD and HBD-1, and HBD-2 levels in plasma and in BAL fluid (BALF) by radioimmunoassay. Plasma concentrations of HAD and HBD-2 in those patients were higher than those in control subjects, whereas HBD-1 levels were similar to those in the control subjects. High levels of HAD and HBD-2, but not HBD-1, also were observed in the BALF of MAI patients. There was a positive correlation between HAD and interleukin (IL)-8 concentrations in the BALF of patients with MAI. BALF HBD-2 concentrations also correlated positively with those of plasma HBD-2 and BALF IL-1 beta in MAI patients. Patients with cavity formation on the chest roentgenogram had higher HAD and HBD-2 levels in their BALF than those of patients without cavity formation. Treatment with clarithromycin combined with two or three other antibiotics, including ethambutol, rifampicin, ofloxacin, or ciprofloxacin, for at least 6 months resulted in a significant fall in plasma HBD-2 concentrations in responders, but not in nonresponders. CONCLUSION: Our findings suggest that HAD and HBD-2 may participate in host defense and local remodeling of the respiratory tract in patients with MAI and that plasma HBD-2 levels may be a useful marker of disease activity in patients with pulmonary MAI.

Selective fluorescent labeling of the 50-, 26-, and 20-kilodalton heavy chain segments of myosin ATPase.

7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin (CPI), rhodamine B isothiocyanate (RITC), and 4-bromomethyl-6,7-dimethoxycoumarin (BDMC), fluorescent reagents that can react covalently with amino or sulfhydryl groups, have been used to label myosin subfragment-1 (S-1) ATPase. The conditions under which CPI, RITC, and BDMC selectively label the 50-, 26-, and 20-kDa segments of the S-1 heavy chain, respectively, are described. CPI and RITC labeling little affects the ATPase activities of S-1 in the presence and absence of actin. BDMC labeling activates the Ca2+- and Mg2+-ATPases of S-1, and abolishes the K+-EDTA-ATPase. The three S-1 derivatives fluoresce strongly even under acidic conditions, suggesting the wide applicability of these fluorescent reagents as selective labels for the three segments of the S-1 heavy chain.

Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin.

In order to obtain information about the actin-induced conformational change around the subfragment-1/subfragment-2 link region of myosin, measurements of the fluorescence quenching by acrylamide were made on cardiac myosin and its heavy meromyosin, in which the reactive lysyl residue located in the link region was labeled with an extrinsic fluorophore, the N-methyl-2-anilino-6-naphthalenesulfonyl group. The results with the model compound indicated the involvement of a collisional quenching mechanism for the fluorophore. The quenching rate constant calculated from measured quenching constants using available lifetime data was extremely low for the labeled myosin (0.59 X 10(8) M-1 . S-1), suggesting that the fluorophore bound to myosin is surrounded by segments of proteins. This value was independent of the solvent viscosity, indicating that the quenching reaction is limited by fluctuations in the protein matrix, which produce the inward movement of acrylamide. Chymotryptic digestion of the labeled myosin, which yielded the light chain-2-deficient heavy meromyosin, made the bound fluorophore slightly exposed. Addition of F-actin resulted in about 40% reduction in the quenching rate constants for the labeled myosin and heavy meromyosin. The actin effect was reversed by adding ATP. These results suggest that the binding of actin to myosin makes the protein matrix around the subfragment-1/subfragment-2 link region less mobile.

2' (or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-triphosphate as a probe for the binding site of heavy meromyosin ATPase.

1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad(, and 1-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule. 2. A broad new absorption band was observed in the 530-630 nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550 nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution. 3. The binding of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 muM and a maximum binding number of 1.8 were obtained in 0.15 M KCl, 10 mM MgCl2, and 50 mM Tris-HCl (pH 8.0) at 25 degrees. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6 M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.

Affinity labeling of the myosin ATPase with ribose-modified fluorescent nucleotides and vanadate.

Ribose-modified fluorescent nucleotide analogs, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives of AT(D)P, dAT(D)P, CT(D)P, UT(D)P, IT(D)P, and GT(D)P, were synthesized for use as substrates and affinity labels for the myosin ATPase [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. None of the fluorescent nucleoside triphosphate (NTP) analogs was significantly different from the corresponding natural NTP in its ability to support superprecipitation of actomyosin. When fluorescent and natural NTPs were used as substrates for the myosin subfragment-1(S-1) ATPase in the presence of 1mM vanadate ion (V1), a slight initial inhibition of the S-1 NTPase was followed by progressive inhibition to more than 60% over a period of 1 h. The apparent second-order rate constants were 0.14-0.44M-1 . s-1, suggesting the formation of the inactive fluorescent NDP-labeled S-1. After incubation of S-1 with the nucleoside diphosphate (NDP) analog in the presence of Vi, the resultant fluorescent NDP-labeled S-1 was isolated free of unbound Vi and the analog by gel filtration. The isolated complexes had stoichiometries of 0.6-1.1 NDP analog per S-1 active site. Native polyacrylamide gel electrophoresis revealed conveniently that the NDP analog is associated with S-1 as indicated by two intense fluorescent bands corresponding to S-1 isozymes. On dissociating gels, the analog was released from S-1, suggesting that the labeled S-1 is held together by strong secondary forces rather than covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct structures of ATP and GTP complexes in the myosin ATPase.

The active site of the myosin subfragment-1 ATPase was affinity-labeled with ribose-modified fluorescent analogs of ADP, dADP, CDP, UDP, IDP, and GDP in combination with vanadate, forming a stable myosin-nucleoside diphosphate-vanadate complex that is analogous to the normal myosin-ADP-Pi intermediate [Hiratsuka, T. (1984) J. Biochem. 96, 147-154]. Labeled enzyme was isolated free of unbound analog and vanadate, and fluorescent properties of the fluorophore at the active site were examined. Fluorescence emission and acrylamide quenching studies revealed that the hydrophobicity of environment around the fluorophore and the degree of its burial in the protein vary with the base structure of NDP. It was found that the fluorophore of ADP analog is most buried into the protein, while that of the GDP analog is least buried. The results suggest that the deep burial of ATP into the myosin active site is essential for muscle contraction.

Direct cross-linking of three domains in the myosin head.

To obtain information about domain-domain contacts in the myosin head, trypsin-split myosin subfragment-1 (S-1), which mainly consists of 50K, 26K, and 20K fragments, was cross-linked directly by the photodynamic technique under mild conditions (pH 7-8.5, 0 degrees C) using riboflavin 5'-phosphate, protoporphyrin, and methylene blue as sensitizers. Exposure of trypsin split-chymotryptic S-1 to visible light in the presence of the sensitizers resulted in formation of cross-linked products of (20 + 26)K, (20 + 50)K, (26 + 50)K, and (20 + 26 + 50)K fragments. The results suggest that three domains in the myosin head are in contact with each other, at least partly, raising the possibility that the communication between the ATP- and actin-binding sites passes through a "short cut," i.e., the contact region between the domains.