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P D Benya

Publications and source records attributed to P D Benya.

At least 19 recordsLinked to original sources
PubMed2001

Characteristics of degeneration in an unstable knee with a coronal surface step-off.

To investigate the effect of instability on the remodelling of a minor articular surface offset, we created a 0.5 mm coronal step-off of the medial femoral condyle in 12 New Zealand white rabbits and transected the anterior cruciate ligament (ACL). A control group of 12 rabbits had only ACL resection and the opposite knee was used as the non-operated control. The osteoarthritic changes at 6, 12 and 24 weeks after surgery were evaluated histologically. In addition, changes in the immunological detection of 3-B-3(-) and 7-D-4 chondroitin-6-sulphate epitopes were determined because of the previous association of such changes with repair of cartilage and early osteoarthritis. In the instability/step-off group there was rapidly progressing focal degeneration of cartilage on the high side of the defect, not seen in previous step-off studies in stable knees. The rest of the femoral condyles and the tibial plateaux of the instability/step-off group had moderate osteoarthritis similar to that of the instability group. 3-B-3(-) was detectable in the early and the intermediate stages of osteoarthritis but no staining was seen in the severely damaged cartilage zones. Immunoreactivity with 7-D-4 increased as degeneration progressed.

Animals↗
PubMed1999

Effect of protein lubrication on the wear properties of materials for prosthetic joints.

The effects of pre-dilution and other modifications of bovine serum lubricants on the wear properties of UHMW polyethylene acetabular cups were evaluated in a hip joint simulator. The wear rate increased, and a nonphysiological type of surface-pitting occurred, when the serum was pre-diluted to 40% or lower concentration. During the wear test, the equilibrium temperature and the precipitation of proteins were substantially greater with zirconia balls than with cobalt-chromium. Protein precipitation, a potential modulator of in vitro wear, was shown to be temperature, concentration, and time-dependent in water-bath tests, which indicated that ball-cup interface temperatures in the simulator must be above 60 degrees C, i.e., well above the bulk lubricant temperature, to account for wear test protein precipitation. Several modifications of serum that were, in part, intended to decrease the tendency for protein precipitation were found to markedly affect the wear properties of the two combinations of materials. In particular, modified serum, which lacked some of the higher molecular weight proteins, produced a much higher wear rate than a control serum with the same initial protein concentration. The results indicated directions for further research to clarify the lubricating properties of serum, and for developing a universal standard test lubricant.

Animals↗
PubMed1998

Metal wear particle characterization from metal on metal total hip replacements: transmission electron microscopy study of periprosthetic tissues and isolated particles.

The less intense tissue reaction around metal on metal total hip replacements (THRs) compared to metal on polyethylene (PE) THRs may be explained by the differences in the characteristics of metal wear particles. In this study, transmission electron microscopy was used to study metal wear particles that were either in situ in cells or had been extracted from the cells by a new technique based on enzymatic tissue digestion. The tissues were obtained from 13 patients undergoing revision of metal on metal THRs with cobalt-chromium-molybdenum (CoCrMo) bearing couples. Most of the CoCrMo wear particles were smaller than 50 nm (range 6-834 nm) and round to oval in shape with irregular boundaries. This size range is considerably smaller than that reported for PE particles. While even a small volume of metal wear will produce high numbers of particles, the apparently less severe local tissue reaction to metal particles may be due to the possibility that corrosion, dissolution, and dissemination of metal particles may result in fewer local biological effects than the long-term retention of PE particles in the periprosthetic tissues.

Adult↗
PubMed1998

Cartilage changes caused by a coronal surface stepoff in a rabbit model.

Coronal stepoffs of 0.5 mm (equal to the cartilage height) were created on the medial femoral condyles of adult, skeletally mature rabbits as a model for articular surface incongruity. After 3, 6, 12, and 24 weeks, tissue was analyzed histologically using hematoxylin and eosin and Safranin O staining, autoradiographs were made of the femoral condyles, and immunohistologic analysis was done for 3-B-3(-) and 7-D-4 chondroitin sulfate epitopes. An overlapping flap from the high toward the low side and an increase of the cartilage height on the low side of the defect were observed as permanent features of adaptation throughout the entire followup. Significant degeneration was not seen around the lesion or in the tibial cartilage opposing a stepoff defect. Autoradiography showed a three-phase response to the lesion: an early increase in radiolabeled sulfate (35SO4) uptake, a sharp decline of 35SO4 uptake, and finally a late recovery of the autoradiographic signal indicating partial recovery of proteoglycan synthetic activity. After an early increase, immunohistologic analysis for 3-B-3(-) showed a subsiding tendency by 24 weeks, and the staining with 7-D-4 remained elevated uniformly in the vicinity of the lesion. A rabbit femoral stepoff defect with an offset of 0.5 mm may remodel and not lead to degeneration within the first 6 months after injury in a stable joint.

Adaptation, Physiological↗
PubMed1996

Transforming growth factor-beta type-II receptor signalling: intrinsic/associated casein kinase activity, receptor interactions and functional effects of blocking antibodies.

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.

Amino Acid Sequence↗
PubMed1996

Surface adhesion-mediated regulation of chondrocyte-specific gene expression in the nontransformed RCJ 3.1C5.18 rat chondrocyte cell line.

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.

Animals↗
PubMed1996

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Aggrecans↗
PubMed1993

Dihydrocytochalasin B enhances transforming growth factor-beta-induced reexpression of the differentiated chondrocyte phenotype without stimulation of collagen synthesis.

Rabbit articular chondrocytes were treated with retinoic acid (RA) to eliminate the differentiated phenotype marked by the synthesis of type II collagen and high levels of proteoglycan. Exposure of such cells to transforming growth factor-beta 1 (TGF-beta 1) in secondary culture under serum-free and RA-free, defined conditions led to reexpression of the differentiated phenotype. The microfilament modifying drug, dihydrocytochalasin B (DHCB), enhanced the effectiveness of TGF-beta 1 and produced a threefold stimulation of type II collagen reexpression (measured by 2-D CNBr peptide mapping) at 0.3 ng/ml TGF-beta 1 without altering total collagen synthesis. Type II collagen reexpression was maximal from 1 to 5 ng/ml TGF-beta 1, with or without DHCB. The effect of DHCB on proteoglycan synthesis was maximal at 1 ng/ml TGF-beta 1. At this dose TGF-beta alone produced no increase in 35SO4 incorporation, while simultaneous treatment with DHCB caused a sevenfold stimulation of proteoglycan synthesis. DHCB-independent stimulation proteoglycan reexpression occurred between 5 and 15 ng/ml TGF-beta 1. In contrast, TGF-beta 1-dependent stimulation of proteoglycan synthesis in differentiated chondrocytes in primary monolayer culture was not substantially affected by DHCB. The collagen data suggest that TGF-beta 1 utilizes separate pathways to control phenotypic change and collagen (matrix) synthesis. Microfilament modification by DHCB selectively enhances the effectiveness of the TGF-beta 1-dependent signaling pathway that controls reexpression of the differentiated phenotype.

Actin Cytoskeleton↗
PubMed1992

SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions.

Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.

Animals↗
PubMed1990

A highly specific and quantitative method for determining type III/I collagen ratios in tissues.

The distribution of type I and type III collagens in rat, bovine and human skin were examined by a quantitative 2-D CNBr peptide mapping method. The procedure involved the solubilization of tissues by digestion with CNBr, radioactive labeling in vitro by [3H]-NaBH4 in dimethylformamide, reduction by mercaptoethanol, a second CNBr digestion and 2-D (isoelectric focusing and NaDodSO4 electrophoresis) mapping. The amounts of type I and type III collagen peptide spots in the fluorographs of 2-D maps were analyzed by 2-D scanning densitometer/analyzer. Mixtures containing various ratios of purified type I and type III collagen were used to obtain a standard curve. Using this procedure we were able to determine that in adult human skin (age range 35-65 years) 22% (+1.3%) of the labelled collagen is type III. This value is significantly higher than that was previously estimated by less accurate methods.

Adult↗
PubMed1989

The cloning and sequencing of alpha 1(VIII) collagen cDNAs demonstrate that type VIII collagen is a short chain collagen and contains triple-helical and carboxyl-terminal non-triple-helical domains similar to those of type X collagen.

We have isolated two overlapping cDNA clones covering 2425 base pairs encoding a short type VIII collagen chain synthesized by rabbit corneal endothelial cells. The cDNAs encode an open reading frame of 744 amino acid residues containing a triple-helical domain of 454 residues flanked by 117- and 173-residue amino and carboxyl non-triple-helical domains (called NC2 and NC1, respectively). Based on the identity between the DNA-derived amino acid sequence and the amino acid sequence of a type VIII collagen CNBr peptide obtained from rabbit corneal Descemet's membrane, we conclude that the cDNAs code for a type VIII collagen chain. We give this chain the designation alpha 1(VIII). The alpha 1(VIII) triple-helical domain contains eight imperfections in the Gly-X-Y repeated structure with Gly-X instead of a full triplet. The length of the triple-helical domain and number and relative locations of these imperfections are remarkably similar to those of chicken alpha 1(X) collagen. The amino acid sequence of the carboxyl three-quarters of the NC1 domain has high sequence similarity to that of alpha 1(X) collagen. These data suggest that the triple-helix coding portions and carboxyl three-quarters of the NC1 domains of the alpha 1(VIII) and alpha 1(X) genes have a common evolutionary origin.

Amino Acid Sequence↗
PubMed1988

Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape.

Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA-modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture.

Actin Cytoskeleton↗
PubMed1988

Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression.

The differentiated phenotype of rabbit articular chondrocytes was modulated in primary culture by treatment with 1 microgram/ml retinoic acid (RA) and reexpressed in secondary culture by treatment with the microfilament-disruptive drug dihydrocytochalasin B (DHCB) in the absence of RA. Because the effective dose of DHCB (3 microM) did not elicit detectable cell rounding or retraction, the nature and extent of microfilament modification responsible for induction of reexpression was evaluated. The network of microfilament stress fibers detected with rhodamine-labeled phalloidin in primary control chondrocytes was altered by RA to a "cobblestone" pattern of circularly oriented fibers at the cell periphery. Subsequent treatment with DHCB resulted in rapid changes in this pattern before overt reexpression. Stress fibers decreased in number and were reoriented. Parallel arrays of long fibers that traversed the cell were evident, in addition to fiber fragments and focal condensations of staining. Immunofluorescent staining of intermediate filaments revealed a marked decrease in complexity and intensity during RA treatment but no change during reexpression. An extended microtubular architecture was present throughout the study. These results clearly identify microfilaments as the principal affected cytoskeletal element and demonstrate that their modification, rather than complete disruption, is sufficient for reexpression. The specificity of DHCB and the reorientation of these filaments before reexpression of the differentiated phenotype suggests a causative role in the mechanism of reexpression.

Actin Cytoskeleton↗
PubMed1986

Coordinate regulation of type IX and type II collagen synthesis during growth of chick chondrocytes in retinoic acid or 5-bromo-2'-deoxyuridine.

Chondrocytes isolated from 15-day-old embryonic chick sterna were cultured as monolayers for 7 days in control medium or in medium supplemented with retinoic acid or 5-bromo-2'-deoxyuridine. Control cells exhibited characteristic polygonal morphology and maintained the synthesis of cartilage-specific collagens, i.e. type II, type IX, 1 alpha, 2 alpha, and 3 alpha chains, and 45 K (presumptive type X). Type IX was the second most prevalent collagen and represented 12-15% of the phenotype. When exposed to retinoic acid, chrondrocytes displayed a fibroblast-like morphology and decreased collagen synthesis by day 2. The synthesis of collagen types II and IX declined in parallel along with that of the other cartilage collagens and ceased by day 7. During the same period, the synthesis of collagen types I, III, and V and two unidentified collagen chains was initiated and stimulated. Similar changes in collagen expression were caused by 5-bromo-2'-deoxyuridine but were delayed, beginning after day 4. Type III collagen, however, was never detected in 5-bromo-2'-deoxyuridine or control cultures. Because two different agents and two rates of modulation produced parallel changes in the synthesis of collagen types II and IX, these collagens appear to be coordinately regulated.

Animals↗
PubMed1986

Isolation and characterization of type VIII collagen synthesized by cultured rabbit corneal endothelial cells. A conventional structure replaces the interrupted-helix model.

Radioactive proline-labeled type VIII collagen was biosynthesized in the presence of beta-aminoproprionitrile by rabbit corneal endothelial cells and isolated from the culture medium. Type VIII was purified in the presence of protease inhibitors and at neutral pH by ultrafiltration, precipitation with 3.9 M NaCl, sedimentation in sucrose gradients, and DEAE-Sephacel chromatography. The major components of this collagen, VIII-1, -2, and -3, exhibited apparent molecular weights of greater than 194,000, 124,000, and 61,000, respectively, and were shown to contain identical CNBr peptides. Following separation of VIII-1, -2, and -3 from each other and any residual proteases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exposure to acetic acid led to the conversion of VIII-1 to VIII-2 and VIII-3. Thus, VIII-1 is not a continuous single peptide chain, and the preliminary interrupted-helix model of the type VIII structure (Benya, P. D. (1980) Renal Physiol. 3, 30-35) was revised. VIII-3 appears to be the parent alpha 1 (VIII)-chain, with VIII-2 and VIII-1 representing beta- and gamma-chain configurations stabilized by strong noncovalent acid-labile interactions and beta-aminoproprionitrile-insensitive covalent cross-links. Based on two-dimensional CNBr peptide mapping, the alpha-chain is composed of six peptides. Mr 5,300-19,600. The terminal peptides are pepsin sensitive and correlate with two noncollagenous domains, NC1 (Mr 14,700) and NC2 (Mr 4-5,000). NC1 contains the site of acid-labile chain association.

Animals↗
PubMed1986

Modulation of the rabbit chondrocyte phenotype by retinoic acid terminates type II collagen synthesis without inducing type I collagen: the modulated phenotype differs from that produced by subculture.

The differentiated phenotype of rabbit articular chondrocytes can be characterized by the synthesis of high levels of cartilage specific proteoglycan and collagen (type II). Treatment of these cells in primary monolayer culture for periods of up to 18 days with 0.03 to 3.0 micrograms/ml retinoic acid (RA) resulted in suppression of colony formation, altered morphology, and decreased (eightfold) proteoglycan and collagen synthesis. With the exception of collagen synthesis, these changes were complete with all doses after 4 days of treatment. Collagen synthesis declined more slowly; it was dose dependent after 4 days and maximally inhibited by all doses by 9 days. Detailed analysis of the collagen phenotype was performed using SDS-PAGE of intact chains and 2-D CNBr peptide analysis. RA caused cessation of type II synthesis, and transient stimulation of type III and type I trimer collagen synthesis, without induction of type I collagen. Essentially identical results were obtained with retinol. The resultant collagen phenotype differed significantly from the type I-containing phenotype induced by subculture. Thus, suppression of this differentiated program did not elicit a common modulated phenotype. The results are discussed in the context of direct and indirect mechanisms of RA-dependent modulation of chondrocyte gene expression.

Animals↗
PubMed1984

Identification of a large interrupted helical domain of disulfide-bonded cartilage collagen.

In order to characterize a larger form of disulfide-bonded cartilage collagen, explants of 17-day embryonic chick sterna were cultured in the presence of [3H] proline. Radioactive collagen chains and fragments that were synthesized and secreted into the culture medium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. After limited pepsin digestion of the medium, two discrete disulfide-bonded collagen fragments were detected with Mr = 210,000 and 153,000. These fragments contained 28 and 17.5%, respectively, of the radioactivity in the alpha 1(II)-chains. The smaller fragment (called M) produced three components upon reduction (Mr = 104,000, 51,000, and 31,000) and seemed to represent the previously reported collagens, HMW and M1. The larger fragment (called N) has not been previously described and gave rise to three components upon reduction (Mr = 140,000, 69,000, and 49,000). Prolonged pepsin treatment resulted in the gradual decrease of N with a corresponding increase of M, suggesting the conversion of N to M. CNBr peptide mapping demonstrated that all M-derived peptides were present in N and that N contained extra peptides that account for its larger size. These observations suggest that N represents a larger more intact form of cartilage-derived disulfide-bonded collagen.

Animals↗