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PubMed1993

Molecular structural changes in Mallory body proteins in human and mouse livers: an infrared spectroscopy study.

To study the molecular structure of Mallory body (MB) proteins we applied infrared spectroscopy of the isolated MBs from livers obtained from autopsied patients with alcoholic cirrhosis and griseofulvin-fed (GF-fed) mice. Liver frozen sections were extracted with detergent and digested with deoxyribo- and ribonuclease and collagenase. MB-enriched fractions were then separated out using the aqueous two-phase polymer system. Immunohistochemical and electron microscopic examination showed that the MB composition was virtually identical in human and mouse livers. Infrared spectra of both MB samples showed that the MBs had more numerous and stronger intermolecular hydrogen bonding than did the background control fractions as well as the cytoskeletal fraction from control and GF-fed mice. This may explain why the proteins in MBs are aggregated. The relative amount of beta-sheets was increased compared to the alpha-helices in the MBs, indicating that conformational changes in the cytokeratin peptides of the MBs had occurred. This may explain why the antigenic sites observed in MB proteins show changes in affinity for antibodies to cytokeratins as observed by immunohistochemical staining of MBs.

Animals↗

PubMed1998

X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP.

To clarify the structure of the ATP hydrolysis intermediates (ADP.Pi bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeFx = 3,4 and AlF4) and vanadate (VO4(Vi)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (approximately 50% decrease in intensity) was observed in the presence of BeFx among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca(2+)-activated fibers, while the first actin layer line at approximately 1/36.7 nm-1 retained a rigor-like profile with an intensity weakened by approximately 50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (approximately 1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeFx. These results indicate that a substantial fraction (approximately 40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeFx bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603-608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.

Adenosine Diphosphate↗

PubMed1998

Fine structural changes in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenic potatoes.

The present work has been designed to study the effect of feeding on transgenic potatoes, which carry the CryI gene of Bacillus thuringiensis var. kurstaki strain HD1, on the light and electron microscopic structure of the mice ileum, in comparison with feeding on potatoes treated with the 'delta-endotoxin' isolated from the same bacterial strain. The microscopic architecture of the enterocytes of the ileum of both groups of mice revealed certain common features such as the appearance of mitochondria with signs of degeneration and disrupted short microvilli at the luminal surface. However, in the group of mice fed on the 'delta-endotoxin', several villi appeared with an abnormally large number of enterocytes (151.8 in control group versus 197 and 155.8 in endotoxin and transgenic-treated groups, respectively). Fifty percent of these cells were hypertrophied and multinucleated. The mean area of enterocyte was significantly increased (105.3 microm(2) in control group versus 165.4 microm(2) and 116.5 microm(2) in endotoxin and transgenic-treated groups, respectively). Several forms of secondary lysosomes or auotophagic vacuoles were recognized in these cells. These changes were confirmed with the scanning electron microscope which revealed a remarkable increase in the topographic contour of enterocytes (23 microm in control group versus 44 microm and 28 microm in endotoxin and transgenic-treated groups, respectively) at the divulged surface of the villi. The basal lamina along the base of the enterocytes was damaged at several foci. Several disrupted microvilli appeared in association with variable-shaped cytoplasmic fragments. Some of these fragments contained endoplasmic reticulum, as well as ring-shaped annulate lamellae. In addition, the Paneth cells were highly activated and contained a large number of secretory granules. These changes may suggest that delta-endotoxin-treated potatoes resulted in the development of hyperplastic cells in the mice ileum. Although mild changes are reported in the structural configuration of the ileum of mice fed on transgenic potatoes, nevertheless, thorough tests of these new types of genetically engineered crops must be made to avoid the risks before marketing.

Animals↗

PubMed2004

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase.

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.

Calorimetry, Differential Scanning↗

PubMed1999

Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding.

The mechanisms of crystalline cellulose degradation by cellulases are of paramount importance for the exploitation of these enzymes in applied processes, such as biomass conversion. Cellulases have traditionally been classified into cellobiohydrolases, which are effective in the degradation of crystalline materials, and endoglucanases, which appear to act on "soluble" regions of the substrate. Humicola insolensCel6A (CBH II) is a cellobiohydrolase from glycoside hydrolase family 6 whose native structure has been determined at 1.9 A resolution [Varrot, A., Hastrup, S., Schülein, M., and Davies, G. J. (1999) Biochem. J. 337, 297-304]. Here we present the structure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 A resolution. Crystals of Cel6A, grown in the presence of cellobiose, reveal six binding subsites, with a single glucose moiety bound in the -2 subsite and cellotetraose in the +1 to +4 subsites. The complex structure is strongly supportive of the assignment of Asp 226 as the catalytic acid and consistent with proposals that Asp 405 acts as the catalytic base. The structure undergoes several conformational changes upon substrate binding, the most significant of which is a closing of the two active site loops (residues 174-196 and 397-435) with main-chain movements of up to 4.5 A observed. This complex not only defines the polysaccharide-enzyme interactions but also provides the first three-dimensional demonstration of conformational change in this class of enzymes.

Binding Sites↗

PubMed1980

Hypoxia-induced structural changes in the media and adventitia of the rat hilar pulmonary artery and their regression.

Chronic hypobaric hypoxia induces structural features characteristic of pulmonary hypertension, but little is known of their reversal. In the present study, rats have been exposed to hypobaric hypoxia for 10 days and subsequently allowed to recover in room air for 3, 14, 28, or 70 days. With the use of 1-mu sections and electron-microscopic and point-counting techniques, the regression of the medial and adventitial changes in the rat hilar intrapulmonary artery has been followed. In the media, 10-day hypoxia causes more than a doubling in thickness due to 1) hypertrophy of smooth muscle cells, particularly of rough sarcoplasmic reticulum and Golgi apparatus; 2) an increase in extracellular connective tissue, microfibrils, collagen fibers, and elastin, and 3) edemalike fluid. In addition, the elestic laminas are doubled in thickness, and myofilamentous processes of the hypertrophied smooth muscle cells contact them. After just 3 days' recovery, some cells have already returned to normal diameter, although medial thickening is unchanged. By Day 14 and at Days 28 and 70 of recovery, medial thickness and cell diameter are within the normal range, and by recovery Day 70, there is a significant increase in the relative areal proportion of extracellular collagen fibers and a decrease in elastin (P less than 0.001). Hypoxia also produces a more than twofold increase in adventitial thickness. Hypertrophy of fibroblasts and an increase in their number contribute to the thickening, as does as increase in collagen fibers. During 3-70 days of recovery, thickness is gradually reduced to normal levels, although it is still significantly above normal at Days 3, 14, and 28. The increases in thickness at these times are due mainly to the accumulation of collagen fibers, which are still apparent after 70 days of recovery. Thus, hypoxia causes a doubling in thickness of the medial and adventitial coats of the hilar muscular pulmonary artery, which with recovery regain near normal thickness but whose structure is altered. The increase in collagen fibers contributes to contracture and reduced distensibility in these vessels, which is apparent in arteriograms as narrowed lumen diameter.

Animals↗

PubMed1994

Brain structure changes in schizophrenics with high serum titers of antibodies to herpes virus.

We compared five indices of brain structure between two groups of schizophrenics, namely, those with high and normal levels of antibody in the serum to herpes virus. Eleven 'immuno-positive' and 21 'immuno-normal' subjects obtained from a concomitant study of serum IgG antibody to viruses underwent magnetic resonance imaging (MRI) utilizing a 1 Tesla magnet and 8 mm thick slices. We measured ventricle-brain ratio (VBR), 3rd ventricle width, cortical atrophy, area of corpus callosum, and frontal lobe area. The differences between groups were assessed by t-test and chi-square analysis. Eight of 11 immuno-positives compared to 7 of 21 immuno-normals showed evidence of cortical atrophy (chi 2 = 4.49, p < 0.03). The immuno-positives had smaller left frontal area (mean + s.d = 125.69 + 21.30 versus 143.76 + 19.84, t = 2.07, p < 0.05) and larger 2nd quadrant of the corpus callosum (mean + s.d. = 1.58 + 0.39 versus 1.27 + 0.52, t = 2.68, p < 0.01). The right frontal area also was smaller in immuno-positives but not significant. VBR, 3rd ventricle and the 1st, 3rd and 4th callosal quadrants did not differ between the groups. We conclude that high antibody titers to herpes found in the sera of some schizophrenics might reflect an earlier pathogenetic process that affected brain development. Further studies of antibodies in CSF and brain structure in these or similar subjects and those suspected to be exposed to viral infections in utero should be vigorously pursued to obtain definitive evidence for this hypothesis.

Adult↗

PubMed2006

Structural changes of cell wall mannans of Candida guilliermondii IFO 10279 strain cells cultured at high temperature.

The morphology, structure, and antigenicity of the cells and the cell wall mannans of the Candida guilliermondii IFO 10279 strain cultivated at 33 and 34 degrees C for 48 h in yeast extract-added Sabouraud liquid medium (YSLM) were compared with those cultivated at 27 degrees C and 33 degrees C and then at 27 degrees C (33-27 degrees C). This strain showed little growth at higher than 35 degrees C. The density of the yeast formed cells decreased, with dry weights of about 50% at 33 and 34 degrees C, and only the cells at 34 degrees C revealed a failure of cytokinesis. The structure of the mannans revealed by (1)H-NMR analysis that the mannans obtained at both 33 and 34 degrees C had drastically decreased two consecutive beta-1,2-linked mannopyranose units at the nonreducing terminal of the alpha-linked oligosaccharides and increased one beta-1,2-linked mannopyranose unit at the nonreducing terminal attached to the alpha-1,3-linked mannose unit and the non-reducing terminal alpha-1,3- and alpha-1,2-linked mannopyranose units. The enzyme-linked immunosorbent assay (ELISA) showed that the mannans obtained at 33 and 34 degrees C had decreased reactivity against the factor serum 9 and increased its reactivity against the factor serum 4, in the commercially available factor serum kit "Candida Check".

Candida↗

PubMed1985

Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering. Signal transmission following effector binding.

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

Adenosine Triphosphate↗

PubMed1982

BCG-induced orchitis: structural changes during the degeneration of seminiferous tubules of rats and rabbits.

The effect of BCG-induced orchitis on the structure of the seminiferous tubules in rats and rabbits was investigated by light and electron microscopy. The formation of cavities between Sertoli cells and the displacement of the cells of the spermatogenic cycle are the earliest changes to be observed. Individual Sertoli cells and the displacement of the cells of the spermatogenic cycle are the earliest changes to be observed. Individual Sertoli cells degenerate and separate from spermatocytes and spermatids. The latter form multinuclear complexes by a broadening of the intercellular bridges. The nuclei of spermatids undergo ring-like chromatin condensation in the rat and swelling in the rabbit. After the loss of spermatocytes and spermatids from the germinal epithelium, the remaining Sertoli cells have a very irregular shape and contain many residual bodies, which are probably derived from previously phagocytosed spermatids. They often contain crystalline inclusions. The nuclei of Sertoli cells show small chromatin condensations. In the rabbit, the tubular wall increases considerably in diameter. In the vicinity of a granuloma in the interstitium caused by BCG inflammatory cells accumulate around the wall of the seminiferous tubules. Although the basal lamina seems to be an obstacle, penetration of macrophages into the tubular lumen could be observed. However, this occurred only after the degeneration of the germinal epithelium.

Animals↗

PubMed1999

A phosphorylation-induced major structural change in the N-terminal domain of the P protein of Chandipura virus.

It has previously been shown that phosphorylation of P protein of vesicular stomatitis virus as well as Chandipura (CHP) virus is required for transcription activation and replication switch. The structural nature of this crucial conformational change, however, is largely unknown. We have studied the phosphorylation-associated conformational change in the P protein of Chandipura (CHP) virus using chemical modification, fluorescence, and circular dichroism spectroscopy. Sulfhydryl groups of unphosphorylated CHP-P protein are unreactive to DTNB under nondenaturing conditions. Upon phosphorylation, one sulfhydryl group becomes reactive. We have identified this sulfhydryl group as cysteine 57. The two tryptophan residues (105 and 135) become significantly more buried in the phosphorylated protein. Circular dichroism spectra show significant enhancement in the far-UV region upon phosphorylation. Anisotropy decay of AEDANS-labeled C57 CHP-P protein shows rapid rotation of the probe, suggesting significant mobility of the N-terminal domain in the phosphorylated P protein. The results suggest a global conformational change in the N-terminal domain of the P protein is induced by phosphorylation and yet the phosphorylated N-terminal domain shows significant flexibility.

Anilino Naphthalenesulfonates↗

PubMed2003

Structural changes in trichocyte keratin intermediate filaments during keratinization.

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.

Animals↗

PubMed1993

Structural changes in the actomyosin cross-bridges associated with force generation.

It is generally thought that to generate active force in muscle, myosin heads (cross-bridges) that are attached to actin undergo large-scale conformational changes. However, evidence for conformational changes of the attached cross-bridges associated with force generation has been ambiguous. In this study, we took advantage of the recent observation that cross-bridges that are weakly attached to actin in a relaxed muscle are apparently in attached preforce-generating states. The experimental conditions were chosen such that there were large fractions of cross-bridges attached under relaxing and activating conditions, and high-resolution equatorial x-ray diffraction patterns obtained under these conditions were compared. Changes brought about by activation in the two innermost intensities, I10 and I11, did not follow the familiar reciprocal changes. Instead, there was almost no change in I11, whereas I10 decreased by 34%. Together with the changes found in the higher-order reflections, the results suggest that the structure of the attached force-generating cross-bridges differs from that of the weakly bound, preforce-generating cross-bridges and possibly also differs from that of the cross-bridges in rigor. These observations support the concept that force generation involves a transition between distinct structural states of the actomyosin cross-bridges.

Actomyosin↗

PubMed1999

Histomorphometric analysis of age-related structural changes in human clitoral cavernosal tissue.

PURPOSE: To characterize age-associated histological changes of human clitoral cavernosal tissue and to determine whether age-related histological changes of clitoral cavernosal tissue correlate with vascular disease-related mortality. MATERIALS AND METHODS: Human clitorises were obtained from 15 fresh cadavers (age: 11 to 90 years) and from 3 patients undergoing clitoral surgery (age: 6 months to 15 years). Cross sections of the clitorises were stained with Masson's trichrome and utilized for computer assisted histomorphometric image analysis to determine the clitoral cavernosal content of smooth muscle and connective tissue. RESULTS: These studies revealed a strong link between increase in age and decreased clitoral cavernosal smooth muscle fibers. In histomorphometry, the percentage of clitoral cavernosal smooth muscle (mean +/- standard error) in an age group of 6 months to 15 years (n = 4) was 65+/-1.5, in 44 to 54 years (n = 7) was 50+/-1.2, and in 55 to 90 years (n = 7) was 37+/-1.3 (ANOVA, p = 0.0001). In the 18 tissues studied, decrease in the percentage of clitoral cavernosal smooth muscle significantly correlated with increase in age (simple regression, r = 0.61). In the age group of 44 to 90 years, clitoral cavernosal fibrosis was significantly greater in the presence of cardiovascular disease-related mortality compared with those without cardiovascular disease-related mortality. CONCLUSION: This study shows that aging women undergo histological changes in clitoral cavernosal erectile tissue. Vascular risk factors may adversely affect the structure of clitoral cavernosal tissue. These findings may be of importance in the pathophysiology of age-associated female sexual arousal disorders.

Adolescent↗

PubMed2005

Effects of ethylene glycol and methanol on ammonia-induced structural changes of the oxygen-evolving complex in photosystem II.

Ammonia is an inhibitor of water oxidation and a structural analogue for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH(3)-specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH(3)-specific binding site on the OEC is estimated to be more than 10 times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH(3)-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations ( approximately 1 M), we conclude that ethylene glycol has a clear effect on the NH(3)-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum. In PSII samples containing 30% (v/v) ethylene glycol, only NH(3), not other bulkier amines (e.g., Tris, AEPD, and CH(3)NH(2)), has a clear effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH(3) and CH(3)NH(2) have a clear effect. On the basis of the results mentioned above, we propose that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH(3) and CH(3)NH(2) to the NH(3)-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% (v/v) methanol does not have any significant effect on the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state g = 2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH(3) upon binding to the Mn site of the OEC that gives rise to the altered S(2) state g = 2 multiline EPR signal.

Ammonia↗

PubMed1985

Structural changes of rat liver microsomal membranes induced by the oral administration of carbon tetrachloride. 31P-NMR and spin-label studies.

The acute effects of carbon tetrachloride (CCl4) on the membrane structure of rat liver microsomes were studied using 31P-NMR and spin-labeling techniques. 31P-NMR spectra of rat liver microsomes were not changed appreciably after the oral administration of CCl4, indicating that the surface structures of microsomal membranes probably are not influenced by the oral administration of CCl4. Four different spin-labeled stearic acids, 5-(N-oxyl-4',4'-dimethyloxazolidine)-stearic acid (5SLS), its methyl ester (5SLSM), 12-(N-oxyl-4',4'-dimethyloxazolidine)-stearic acid (12SLS) and its methyl ester (12SLSM), were used for the estimation of membrane fluidity. The apparent rotational correlation time of 12SLS decreased from 4.0 nsec to 3.0 nsec after the oral administration of CCl4, while the order parameter of 5SLS did not change. The results suggest that CCl4 or its metabolites increase the membrane fluidity of liver microsomes primarily at hydrophobic regions rather than at the surface layer. The ESR spectrum of 5SLSM in microsomal membranes comprised two different signals; one was an anisotropic signal and the other was a rather isotropic one. The ratio of the anisotropic signal to the isotropic one decreased markedly after the oral administration of CCl4 and depended on the dose of CCl4. The suitability of this ESR technique with 5SLSM for the estimation of membrane damage is discussed.

Administration, Oral↗

PubMed1995

Reversible structural changes of octacalcium phosphate and labile acid phosphate.

Acid phosphate is one of the major impurities incorporated into bioapatites, and its quantity and environment in forming mineral have been used as diagnostic probes to pursue acidic precursor(s). Currently, little is known about the structural feature of nonstoichiometric octacalcium phosphate (OCP), which has been advocated to be, most plausibly, mineral salt initially formed during amelogenesis. In the present report, we attempt to define the state of acid phosphate in OCP crystals which were Ca-deficient and contained 40% total phosphate as acid phosphate. We assessed fractions of acid phosphate in discrete environments by extracting the crystals in either deionized water, 10 mmol/L NaOH solution (initial pH 11), or 150 mmol/L Tris buffer at pH 7.4. Solid samples before and after the treatments were examined by chemical analyses and x-ray diffraction. The results indicated that successive extractions with use of the alkaline solution brought about a reversible change (not hydrolysis) in the interior structure of OCP, which accompanied a marked decrease in acid phosphate. A substantial part of the lost acid phosphate was restored during subsequent treatments at neutral pH, and, intriguingly, this restoration accompanied a re-ordering of OCP structure. The data suggested that the acid phosphate in OCP is separated into three pools: (a) a stable pool corresponding to roughly 50 to 60% of the total acid phosphate, (b) a reversibly exchangeable pool corresponding to 25 to 30% of the acid phosphate which may exist either in the water layer or on crystal surfaces, and (c) an unstable (or irreversibly lost) pool corresponding to 15 to 20% of the acid phosphate, a part of which might be explained by the presence of excess hydrogen in OCP. The present work supports the concept that protons and, to a lesser magnitude, phosphate species can diffuse into and out of the OCP lattice prior to initiation of its hydrolytic transition into apatite.

Apatites↗

PubMed1989

Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP.

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.

Actin Cytoskeleton↗