Gerald F. Elliott

X-ray diffraction studies on skinned single fibres of frog skeletal muscle

The myofilament lattice of single fibres of frog semi-tendinosus muscle was studied as a function of sarcomere length in intact fibres (with sarcolemma) and skinned preparations (without sarcolemma); the 1,0 transverse spacing of the filament lattice (d) was measured by X-ray diffraction, and the sarcomere length (S) by light diffraction. When the fresh intact fibre (immediately after the dissection) was stretched, the spacing d decreased so as to maintain d2S (which is in proportion to the lattice volume) at a constant value (2.98 × 109 A3). The filament lattice of intact fibres swelled by 24% over four hours following the dissection. These “old” fibres, however, still maintained a constant volume when the sarcomere length was changed. The lattice volume increased by 28 to 98% (depending on the sarcomere length) when the sarcolemma was removed from the fresh single fibre in a relaxing solution containing chloride as the major anion. The skinned fibre did not behave isovolumically; d2S increased from 3.82 × 109 A3 to 5.82 × 109 A3 as the sarcomere length was changed from 2.0 μm to 3.5 μm. Changing the pH from 7.0 to 6.0 had no effect on the lattice spacing. Reducing the ionic strength from (0.17 to 0.10 did not change the 1,0 spacing at shorter sarcomere lengths (S< 2.7 μm), but increased the spacing slightly (up to 6%) at longer sarcomere lengths (S ≥ 2.7 μm). Experiments were also made with skinned fibres in relaxing solutions containing propionate or sulphate as the major anion, and the filament lattice did not behave isovolumically in either of these solutions. It is concluded that the sarcolemma is necessary to maintain a constant lattice volume in the frog skeletal fibre, and an explanation is given for the mechanism of the constant-volume behaviour of the filament lattice, using Donnan and osmotic equilibria across the sarcolemma.

The organisation of collagen fibrils in the human corneal stroma: A synchrotron X-ray diffraction study

The low angle equatorial diffraction pattern from the human corneal stroma shows that the collagen fibrils have two preferred orientations: inferior-superior and medial-lateral. We have not observed this effect in any other animal species. This arrangement, which was found to be more pronounced in the posterior than in the anterior stroma, was maintained until the last 1 to 2 millimetres before the limbus at which point uniaxial orientation was observed along the circumference. Our interpretation of this result is that most collagen fibrils wrap around the circumference of the cornea and relatively few continue radially into the limbus where uniformity of collagen fibril diameters is lost.

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.

Synchrotron X-ray diffraction study of corneal stroma

Abstract Using a synchrotron X-ray source, it has been possible to record a low-angle diffraction pattern from fresh bovine corneal stroma. The pattern can be interpreted as arising from the short-range order packing of collagen fibrils in lamellae. Model calculations suggest that the positions of the fibrils remain correlated over distances corresponding to, at most, three fibril diameters (~ 120 nm). These results support theories of transparency of the cornea based on short-range order. Further, a study of the fibril spacing as a function of hydration confirms that water uptake occurs largely between the lamellae and in regions devoid of collagen fibrils, and shows that the fibril diameter increases with hydration.

X-ray diffraction studies of the corneal stroma.

Abstract A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling. The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.

Interpretation of the meridional

Abstract The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae. Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found. A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.

X

Abstract Using synchrotron X-ray diffraction we have measured the mean centre-to-centre spacing between the collagen fibrils in the corneal stroma from a variety of vertebrate species. Our observations indicated that the species fall into two main groups: the bony fish, fin whale and dolphin with spacings less than 52 nm, and the other mammals with spacings in the range 59–73 nm. In addition, individual mammalian corneas were shown to contain a variation in fibril spacings which was found to be species dependent. The coypu, rat, guinea pig and squirrel were not typical in that they contained a larger range of spacings than found in the other animals. In a few cases, we have measured both the anterior and posterior sections of corneas; these measurements do not show any significant difference in interfibrillar spacings.

-ray diffraction pattern from collagen fibrils in corneal stroma

We derive the step-size distance, and the impulse time per ATP split, from a consideration of Hills energy rate equation coupled with the enthalpy available per ATP split. This definition of step-size distance is model-independent, and is calculated to have a maximum of 17 A at no load and to reduce to zero at isometric tension, since it will depend on the velocity of shortening. We revisit a derivation of Hills force-velocity equation depending on impulsive forces working against frictional forces and show that this gives a physical meaning to Hills constants a and b. This is particularly elegant for Hills constant b, which is directly related to the impulse time; the value of this impulse time is 1/2 ms. The question that muscle contraction may involve overlapping interactions is considered. However, we find that the step-size distance is not dependent on the possibility of overlapping interactions.

Collagen interfibrillar distances in corneal stroma using synchrotron X-ray diffraction: a species study

Low angle synchrotron X-ray diffraction patterns were obtained from the demembranated human corneal stroma. These patterns showed differences in the relative intensities of the meridional reflections compared to those obtained using bovine corneas. In particular, the first order reflection, conspicuously absent in the bovine pattern, is present in the human, although it is still only one third as intense as the first order in the pattern from rat tail tendon. Using phases deduced from the electron-optical images of negatively stained corneal collagens, a difference electron density map between bovine and human corneal collagen was drawn. The features in this map are explained in terms of a different inter-relationship between collagen and proteoglycans in the two species. The function of these species differences is unknown.

Muscle contraction: the step-size distance and the impulse-time per ATP

A decrease in the net fixed electric charge in the A-bands of cross-striated muscle was observed by Bartels and Elliott [2,10] when the muscle went from the rigor to the relaxed condition. The current work localises the source of the charge decrease by following the net charge on myosin (in the form of concentrated gels) and also myosin rod and light meromyosin gels when the gels are exposed to different concentrations of ATP. The work includes a study of muscle A-bands when the muscle is exposed to the same variations in ATP concentrations as the protein gels. The work shows that (i) Only 100-200 microns ATP is needed to initiate the charge decrease between the rigor and relaxed conditions; (ii) the effect of ATP is seen in the muscle A-band and the myosin and myosin rod gels, but not in LMM gels; (iii) pyrophosphate (PPi) shows a similar charge effect to ATP. ADP does not affect the charge on myosin gels, on the other hand. The results suggest that the charge decrease caused by ATP or PPi is due to ligand interaction with one or more sites on the myosin molecule. This interaction causes a disseminated effect in the protein, and a consequent loss in net negative charge either by a decrease in the absorption, of anions to Saroff sites on the protein, or, less probably, by an increase in the absorption of cations at those sites.