Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

X-ray diffraction studies of the corneal stroma.

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.     

An energetic evaluation of a “Smith” collagen microfibril model

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro) _n , (Gly-Prop-Hyp) _n , or (Gly-Ala-Ala) _n , wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)₁₂ has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)₁₂, (Gly-Pro-Hyp)₁₂, and (Gly-Ala-Ala)₁₂ show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.     

An energetic evaluation of a "Smith" collagen microfibril model.

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro) _n , (Gly-Prop-Hyp) _n , or (Gly-Ala-Ala) _n , wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)₁₂ has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)₁₂, (Gly-Pro-Hyp)₁₂, and (Gly-Ala-Ala)₁₂ show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Studies on the nature of the Z-discs in skeletal muscle fibres of sharks, Etmopterus spinax L. and Galeus melastomus Rafinesque-Schmaltz

The ultrastructural details of Z-discs from red, intermediate, and white axial muscle fibres from the sharks Etmopterus spinax and Galeus melastomus are described. Red fibre Z-discs contain the most amorphous matrix material, and are thicker (100–115 nm) than intermediate (85–88 nm) and white Z-discs (75–80 nm). Four sets of oblique bars extend tangentially from each thin filament. In each set two bars are present, those of white fibres are close together (approximately 5 nm), while those of red fibres are separated by a distance of 15 nm. A model of shark Z-disc structure is proposed. The denaturation (heat transition) temperatures of the muscle proteins were studied by differential scanning calorimetry (DSC). The heat transitions of collagen, actin, and myosin were identified; the actin heat transition temperature increased in the sequence red, intermediate, and white. The total protein pattern of red and white muscle were studied by SDS electrophoresis. A protein with a molecular weight of about 55000 may represent a Z-disc protein.     

Hydration and distance dependence of intermolecular shearing between collagen molecules in a model microfibril

In vertebrates, collagen tissues are the main component responsible for force transmission. In spite of the physiological importance of these phenomena, force transmission mechanisms are still not fully understood, especially at smaller scales, including in particular collagen molecules and fibrils. Here we investigate the mechanism of molecular sliding between collagen molecules within a fibril, by shearing a central molecule in a hexagonally packed bundle mimicking the collagen microfibril environment, using varied lateral distance between the molecules in both dry and solvated conditions. In vacuum, the central molecule slides under a stick-slip mechanism that is due to the characteristic surface profile of collagen molecules, enhanced by the breaking and reformation of H-bonds between neighboring collagen molecules. This mechanism is consistently observed for varied lateral separations between molecules. The high shearing force (>7 nN) found for the experimentally observed intermolecular distance (≈1.1 nm) suggests that in dry samples the fibril elongation mechanism relies almost exclusively on molecular stretching, which may explain the higher stiffnesses found in dry fibrils. When hydrated, the slip-stick behavior is observed only below 1.3 nm of lateral distance, whereas above 1.3 nm the molecule shears smoothly, showing that the water layer has a strong lubricating effect. Moreover, the average force required to shear is approximately the same in solvated as in dry conditions (≈2.5 nN), which suggests that the role of water at the intermolecular level includes the transfer of load between molecules.     

New insight into the shortening of the collagen fibril D-period in human cornea

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066–.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.     

Morphology of sheet-like assemblies of pN-collagen, pC-collagen and procollagen studied by scanning transmission electron microscopy mass measurements

At high concentrations, type I pN-collagen, pC-collagen and procollagen (the first 2 generated from procollagen by enzymic cleavage of C-propeptides and N-propeptides, respectively) can all be made to assemble in vitro into thin D-periodic sheets or tapes. Scanning transmission electron microscopy mass measurements show that the pN-collagen sheets and procollagen tapes have a mass per unit area corresponding to that of approximately 6.8 monolayers of close-packed molecules. pN-collagen sheets are extensive and remarkably uniform in mass thickness (fractional S.D. 0.035); procollagen tapes are neither as extensive nor as uniform in thickness. The mean thickness of pC-collagen tapes is less and the variability is greater. In pN-collagen sheets, the overlap: gap mass contrast in a D-period is increased from 5:4 (the ratio in a native collagen fibril) to 6:4, showing that the N-propeptides do not project into the gap but are folded back over the overlap zone. Assuming all N-propeptides to be constrained to the two surfaces of a sheet, their surface density can be found from the mass thickness of the sheet. In a lateral direction (i.e. normal to the axial direction where the spacing is D-periodic), the N-propeptide domains are calculated to be spaced, centre to centre, by 2.23 (+/- 0.1) nm on both surfaces. This value (approx. 1.5 x the triple-helix diameter) implies close-packing laterally with adjacent domains in contact. Sheet formation and the "surface-seeking" behaviour of propeptides can be understood in terms of the dual character of the molecules, evident from solubility data, with propeptides possessing interaction properties very different from those displayed by the rest of the molecule. The form and stability of sheets (and of first-formed fibrils assembling in vivo) could, it is suggested, depend on the partially fluid-like nature of lateral contacts between collagen molecules.     

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.     

Sequential assembly of collagen revealed by atomic force microscopy.

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.     

Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.     

Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.     

Binding of soluble type I collagen molecules to the fibroblast plasma membrane.

Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.     

Interpretation of the temperature-dependent color of blue copper protein mutants

The electronic absorption spectrum of the mutant of the blue copper protein amicyanin with a pseudoazurin loop (AmiPse) shows a remarkable temperature dependence. The absorption band at approximately 460 nm increases at low temperature while the transition at approximately 600 nm is not much affected by a variation of the temperature. An approximate density functional theory (DFT) study of the active site model [Cu(II)(imidazole)(2)(SCH(3))(S(CH(3))(2))](+) (protein backbone and solvation neglected) leads to two local minimum structures (axial and rhomb) which both have a geometry close to that typical for blue copper proteins. One (rhomb) has two structurally different histidine donors, and this geometry is also found in most experimental type 1 structures. The two forms axial and rhomb are distortional isomers and are energetically almost degenerate. The temperature dependence of the spectrum of AmiPse is interpreted with a temperature-dependent change of the relative population of the two local minimum structures with slightly different energy. The 460 nm transition is believed to be due to preferential population of the structure rhomb; this is in agreement with the published assignment of the high energy transition, based on thorough spectroscopic and computational studies. Consequences of a perturbation of the "gas phase" structures axial and rhomb by the protein and solvation are also discussed on the basis of published, experimentally observed structures and spectroscopic data.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.