A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.
Aims/Background-The morphological changes in Bruch's membrane and its constituent collagen seen Conclusion-Changes in the constituentcollagens may contribute to the accumulation ofdebris in Bruch's membrane with age and interfere with the function of the retinal pigment epithelium, with subsequent consequences for the overlying photoreceptors. (Br_J Ophthalmol 1995; 79: 944-952) The high incidence of age-related macular degeneration (ARMD) and its significant impact on the visual function in the elderly population has led to considerable interest in the function and relations of the photoreceptors, the retinal pigment epithelium (RPE), and its adjacent structures, in particular Bruch's membrane. Their failure to function normally is critical in the pathological process that leads to the development of ARMD, and the changes that occur in these tissues with age and in the disease process are of relevance to our understanding of ARMD.In aging and ARMD considerable morphological changes are seen in Bruch's membrane. Changes include the deposition of material between the RPE and its basement membrane. Drusen, which are focal, lie between the RPE basement membrane and the inner collagenous zone of Bruch's membrane. 1-3 More diffuse changes are also seen, with the deposition of debris, vesicular material, and membrane coated bodies within Bruch's membrane, especially in the inner collagenous zone.4 This material has a high lipid content but its composition appears to vary between individuals.5 It is believed to originate in the photoreceptor outer segments and to be the product of RPE metabolism.69 Although the morphological changes that occur in aging and their nature are well described, the mechanism by which the material is retained in the matrix of the membrane is not understood, nor are the consequences of this deposition process. 10The structure of Bruch's membrane depends on the integrity of collagen fibres and concurrent with the macroscopic changes there appear to be age-related changes in the nature of the collagen. There is an increased number of striated collagen fibres present in both the inner and outer collagenous layers with increasing age. This 64 nm banded material is assumed to be the fibrillar type I collagen. This is accompanied by the deposition of short segments of a 100 nm banded form which is assumed to be collagen type VI.2 8 11 The distribution of the collagen types in Bruch's membrane has been studied with immunohistochemical techniques12 13 which demonstrated the presence of types I, III, IV, and V collagen. The staining for types IV and V appeared to be bilaminar, below the RPE and around the choriocapillaris, while that for types I and III tended to be diffuse throughout the thickness of the membrane. In older tissue there appeared to be increased immunostaining for type I collagen as well as frequent interruptions in the bilaminar staining pattern for types IV and V collagen. Significantly, these interruptions were not always associated with drusen.12 Electron microscopic s...
The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction [Coates, Criddle & Geeves (1985) Biochem. J. 232, 351-356]. In the first step actin is weakly bound and the second step involves the complex isomerizing to a more tightly bound state. This isomerization can be followed specifically by monitoring the fluorescence of actin that has been covalently labelled with N-(pyren-1-yl)-iodoacetamide at Cys-374 [Geeves, Jeffries & Millar (1986) Biochemistry 25, 8454-8458]. We report here that the presence of nucleotides and nucleotide analogues affects the equilibrium between the strongly bound and weakly bound states (referred to as K2). In the presence of ATP, [gamma-thio]ATP or ADP and vanadate a value of approx. less than 10(-2) was estimated for K2. In the presence of PPi or ADP a value of approx. 2.3 or 10 respectively was obtained. An increase in KCl concentration or the presence of 40% ethylene glycol was found to decrease K2 in the presence of ADP. The data presented here are consistent with the two-step binding model proposed by Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell Motil. 5, 351-361], where it was suggested that the transition between weakly bound and strongly bound states is closely associated with the force-generating event in whole muscle.
The adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) induced dissociation of actomyosin subfragment 1 (S1) has been investigated by monitoring the light scattering changes that occur on dissociation. We have shown that ATP gamma S dissociates acto-S1 by a mechanism similar to that of ATP but at a rate 10 times slower. The maximum rate of dissociation is limited by an isomerization of the ternary actin-S1-nucleotide complex, which has a rate of 500 s-1 for ATP gamma S and an estimated rate of 5000 s-1 for ATP (20 degrees C, 0.1 M KCl, pH 7.0). The activation energy for the isomerization is the same for ATP and ATP gamma S, and both show a break in the Arrhenius plot at 5 degrees C. The reaction between acto-S1 and ATP was also followed by the fluorescence of a pyrene group covalently attached to Cys-374. We show that the fluorescence of the pyrene group reports the isomerization step and not actin dissociation. The characterization of this isomerization is discussed in relation to force-generating models of the actomyosin cross-bridge cycle.
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