Cartilage Extracts Research Articles

Collagen glucosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage.

A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.

Aggregation of Cartilage Proteoglycans Extracted with Lanthanum Chloride

Large amounts of proteoglycan can be extracted from bovine nasal cartilage by stirring slices of the tissue in certain high-ionic-strength salt solutions (Sajdera & Hascall, 1969). After removal of most of the salt by dialysis, the proteoglycan can be isolated from the extract by CsCl density-gradient centrifugation (Sajdera & Hascall, 1969). This proteoglycan appears to be a macromolecular aggregate and can be dissociated in 4~-guanidine. Thus, a further CsCl centrifugation of the proteoglycan in the presence of 4 . 0 ~ guanidine allows the separation of a ‘proteoglycan subunit’ and the factors that promote its aggregation (Hascall & Sajdera, 1969). Present evidence suggests that these factors are of two types, a ‘glycoprotein link-factor’ and hyaluronic acid (Hascall & Sajdera, 1969; Hardingham & Muir, 1973; Gregory, 1973). We have previously shown that proteoglycan can be extracted from slices of bovine nasal cartilage by stirring it in 0.5w-LaC13 (Mason & Mayes, 1973). Moreover on diluting the extract to 0.05~-L,aCI,, the solubilized proteoglycan precipitates. A ‘proteoglycan subunit’ fraction can be prepared from the precipitate and its composition closely resembles that of ‘proteoglycan subunit’ fractions isolated from more conventional extracts of the tissue with 2.0M-CaC12 or 4.0w-guanidine hydrochloride (Mayes et al., 1973). Since LaClj probably solubilizes proteoglycan from cartilage by a similar mechanism to other high-ionic-strength salt solutions (see Mason & Mayes, 1973) it seemed likely that the lanthanum extract would also contain aggregation factors. We report below a simple method for isolating a fraction from 0.5 M L ~ C ~ , cartilage extracts which promotes proteoglycan aggregation. The method does not require prolonged density-gradient centrifugation and can be used for large-scale preparations.

Complex formation between lysozyme and cartilage proteoglycans

Density-gradient centrifugation of dissociative extracts of cartilage yields a proteoglycan which forms an ionic complex with lysozyme, a natural component of cartilage whose function in that tissue is unknown. The progress of the reaction is monitored by assay of the supernatant hexuronate content after addition of the precipitant. The lysozyme-proteoglycan complex is solubilized by addition of salts or by alkalinization, but the salt concentration required is greater than that needed to disperse a complex of lysozyme and chondroitin sulfate. Since it appears that the chondroitin sulfate portion of the proteoglycan molecule participates in complex formation with lysozyme, the charge distribution in the proteoglycan probably differs from that in the linear glycosaminoglycan. Cartilage lysozyme could exist in vivo as part of an ionic complex involving proteoglycans, but on a stoichiometric basis, it is unlikely that lysozyme plays a major role in maintenance of the structural integrity of the tissue.

Neutral proteases and cathepsin D in human articular cartilage.

Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.

Neovascularization in the rabbit cornea after intracorneal injections of cartilage extracts

Frozen sections were prepared from articular cartilage from calves. Layers containing intracartilaginous vessels were separated from adjacent layers without vessels. Extracts were prepared and injected injeccorneally in rabbits. Peripheral injections gave inconclusive results. Repeated injections of 0·010–0.15 ml in the centre of the cornea produced heavy vascularization in the eye which was injected with homogenates from cartilage zones not containing vessels. There was only slight to moderate vascularization in the eye which was injected with homogenates from zones containing vessels. Vascularization proceeded through an apparently clear periphery of the cornea. The results were unequivocal in a group of eight rabbits. There was no vascularization in controls injected with saline and no or slight vascularization in controls injected with a papain solution. Furthermore, papain digests from cartilage did not produce any vascularization. The experiments lend support to the existence of a factor stimulating vascular ingrowth in the cornea and present in vessel-free articular cartilage from calves, and further suggest that the factor is a chemically-defined substance, possibly a protein.

Diminished carbonyl reactivity, a biochemical abnormality in aminonucleoside-nephrotic rat skin α- and ß-collagen

Comparative study of chromatographically purified α- and ß-collagen fractions of normal and aminonucleoside-nephrotic rat skin collagen reveals that both the rate and extent of reactivity of such fractions with the highly specific and sensitive carbonyl reagent, N-methyl-benzothiazolone hydrazone hydrochloride (MBTH), are diminished in skin collagen from the aminonucleoside-treated rats. Mechanistically this might be explained either by (a) an inhibitory effect of aminonucleoside on the initial step in the formation of a type of collagen cross-linking, involving enzymatically catalyzed oxidative deamination of the ϵ-amino group of lysyl and/or hydroxylysyl residues in the collagen chains to their respective semi-aldehyde residues; or (b) by direct interaction of aminonucleoside with the semi-aldehyde groups, thus effectively reducing their reactivity with MBTH. Elimination of the latter from consideration would seem to be indicated, however, by the absence of any of the identifying spectral characteristics of the aminonucleoside in skin α- and ß-collagen of aminonucleoside-treated rats, as well as in spectra of exhaustively dialyzed normal rat skin α-collagen which had been incubated for several hours with aminonucleoside. Correlation of the diminished carbonyl reactivity with possible inhibitory effects of aminonucleoside on a specific skin lysyl oxidase activity has not yet been accomplished. Evidence suggesting in vitro inhibitory effects of aminonucleoside on thick embryo cartilage lysyl oxidase activity has been obtained, however, by a modified monoamine oxidase assay. Incubation of cartilage extracts, which contain lysyl oxidase activity (demonstrated by tritium release from [6- 3H]-elastin substrate), with 10 −3 M aminonucleoside prior to assay, in a system utilizing lysine-vasopressin as a substrate, resulted in a considerable reduction in the production of hydrogen peroxide, measured by a sensitive fluorometric procedure.

Saline cartilage extract for healing experimental long bone defects

Saline cartilage extract for healing experimental long bone defects

Proteoglycans of the knee-joint cartilage of young normal and lame pigs.

Intensive rearing, and restricted activity, induce rapid growth in pigs, but they often become lame. Groups of normal and lame pigs reared intensively were killed when 10 or 25 weeks old. Although there were no differences in the overall composition of the knee-joint cartilage of lame and sound animals, the proteoglycans in the cartilage of the lame pigs were extracted more easily by a standardized sequential procedure and contained a higher proportion of molecules of smaller size as assessed by gel chromatography on 6% agarose and Sepharose 4B. These increased at the expense of both the larger and mediumsized molecules. Differences were most evident at 10 weeks of age, when there was twice as much of the smaller proteoglycans in the cartilage of lame pigs. Despite these size-differences, the compositions of the proteoglycans in corresponding sequential extracts of cartilage of lame and normal groups were the same, as were the changes in chemical composition that accompany development. Proteoglycans from lame animals may have undergone limited proteolysis, thus decreasing their size without changing their composition detectably. As the differences between normal and lame groups were greater at 10 weeks than at 25 weeks of age, the first weeks after birth (when the greatest changes occur in the proteoglycans and in the cartilage) may be a critical period in the maturation of articular cartilage in this species. At this time, rapid gain in weight produced by intensive rearing may be too great for the immature cartilage to bear.

Endogenous steroid sulfation on incubation of tissue extracts with [ 35S]sulfate

Steroid [ 35S]sulfates can be formed from endogenous acceptors upon incubation of certain tissue extracts with [ 35S] sulfate, or adenosine-3′-phosphate-5′-phospho[ 35S] sulfate of high specific activity. These radioactive components may possess abnormal paper Chromatographic properties and addition of steroid to the incubations can specifically increase the radioactivity therein without giving rise to a product in the expected position for authentic non-labeled steroid sulfate. Addition of the latter as carrier prior to chromatography overcomes this R F variation, suggesting that the abnormal Chromatographic properties are caused by the employment of ‘sub-chemical’ concentrations. The steroid sulfate formed from endogenous steroid in the case of embryonic chick cartilage extracts has been identified as estrone sulfate.

A Double Blind Trial with Cartilage and Bone Marrow Extract in Degenerative Gonarthrosis

SummaryA double-blind trial with cartilage and bone-marrow extract (Rumalon A) and placebo (Rumalon B) was carried out on 106 patients with degenerative bilateral gonarthrosis. Favorable results were obtained in 64 % of the patients treated with Rumalon A and in only 29 % of the placebo group. This difference is statistically significant (p <0.05). The slight influence of placebo can be related to the psychosomatic effect of injections and/or coincident spontaneous improvement of the gonarthrosis. Cartilage and bone marrow extract increases the therapeutic possibilities in degenerative joint disease. It is easy to administer and practically without side effects.

Involvement of UDP derivatives in the transformation of [ 14C]glucose into chondroitin sulphate by extracts of embryonic cartilage

Involvement of UDP derivatives in the transformation of [ 14C]glucose into chondroitin sulphate by extracts of embryonic cartilage

The protein-polysaccharides of articular, epiphyseal plate and costal cartilages

1. 1. The yields of protein-polysaccharides obtained from bovine fetal articular, epiphyseal plate and porcine costal cartilage and their content of uronic acid, hexosamine, sialic acid, protein hydroxyproline were determined. The ability of the protein-polysaccharides to inhibit the precipitation of calcium phosphate in vitro was also studied, both before and after degradation of the protein-polysaccharides by enzymes extracts of epiphyseal plate cartilage. 2. 2. Yields and compositions of protein-polysaccharides from the diverse hyaline cartilages were similar, indicating that there are, in the narrow age range studied, in the different cartilages and in the different species of animals, remarkable similarities in the chemical nature of the hyaline cartilages with respect to their content of protein-polysaccharides. Differences in composition are manifest, however, when compared with calf nasal cartilages or nucleus pulosus. 3. 3. Most of the protein-polysaccharides inhibited the precipitation of calcium phosphate from solution. If the protein-polysaccharides are first degraded by enzyme extracts obtained from cartilage, the inhibitory activity of the protein-polysaccharides is abolished in the range of concentrations studied. These phenomena are discussed in relation to calcification of cartilage.

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo.

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.

Cartilage extract in treatment of fractures in rabbits.

Cartilage extract in treatment of fractures in rabbits.

Cartilage extracts in the treatment of delayed union.

EXPERIENCE has shown that cartilage and its extracts have a favorable influence on soft tissue healing. 1 Trials with similar products in the local treatment of experimental bone defects are associated with such complications as abscess formations. 2 Nevertheless, in those cases where no untoward local reaction is observed, bone healing seems somewhat accelerated. An exclusively parenteral treatment with cartilage extracts might reduce the incidence of complications. We tested the possible effect of such a treatment in cases in which spontaneous union would be delayed. The present paper reports on a series of rabbits and guinea pigs with experimentally induced delayed bone union treated with parenteral saline cartilage extracts. Material and Methods Rabbits. —Eleven adult female rabbits, weighing, 4 ∓ 0.2 kg each, were operated on and a 3 mm segment was bilaterally removed from the midshaft of the radius. The bone ends on both sides of the removed

Stimulation of protein--chondroitin sulfate synthesis by normal and osteoarthritic articular cartilage.

AbstractA stimulatory effect on 35S uptake of rat costal cartilage by a commercial cartilage and bone marrow extract known as Rumalon was confirmed. In addition, the preparation was found to increase35S incorporation in vitro into chondroitin sulfate by human articular cartilage from the knee joint, both normal and osteoarthritic sites. This effect was limited to young rats, but no age limitation in effect was found with human cartilage. In other in vitro studies, the augmented 35S incorporation into chondroitin sulfate was found in fractions eluted from Dowex‐1 columns; a stimulation of incorporation of 3H‐serine into protein‐polysaccharide was also found in the same fraction. The effects of 3H‐serine and 35S uptake were augmented by addition of amino acids to the incubation medium and were inhibited by puromycin. In addition, increased incorporation of 3H‐uridine into RNA was observed in the same system.

Rivanolによるコンドロイチン硫酸改良比濁定量法

The turbidimetric method for determination of chondroitin sulfate (CSA) with rivanol has been proposed by NÉMETH-CSÓKA. When CSA in the extracts of the whale nasal cartilage with 5% Na2CO3 was determined by this original method, the values were lower than those obtained by carbazol method (Table 2). In this paper, the cause was elucidated and the author established a modified method. Chondromucoprotein (CMP), in which CSA is combined with “protein core” and therefore could be regarded as “combined CSA”, was prepared from the whale nasal cartilage by water extraction and “free CSA” from CMP. It was found that rivanol turbidity produced with “combined CSA” was much lower than that produced with “free CSA” (Fig. 1). As it is known that CMP is easily degraded into CSA and “ protein core” by alkali treatment, the effect of NaOH treatment of CMP on rivanol turbidity was examined (Fig. 2). As the results, in determining CSA in CMP by rivanol turbidity method, it is necessary to treat the sample with 1% NaOH for 3 hours at 25°C for degradation of CMP, as a pretreatment. Determination of CSA in mixtures of “free CSA” and “combined CSA”, which were prepared from CSA and CMP, by this modified rivanol method gave a satisfactory result (Table 3).

Hydroxylation of proline after the release of proline-rich polypeptides from ribosomal complexes during uninhibited collagen biosynthesis

Pulse-labeling techniques were used to study the hydroxylation of proline in isolated embryonic cartilage which synthesizes collagen at a rapid rate in vitro. In cartilage incubated with [ 14C]proline for short periods of time, the synthesis of [ 14C]hydroxyproline consistently lagged behind the incorporation of 14C into protein. The ratio of [ 14C]hydroxyproline to total 14C in non-dialyzable fractions was less than 1 % after incubation with [ 14C]proline for 1 min, and it gradually approached a maximal value of 20 to 25 % in 10 to 30 min. Incubation of extracts of pulse-labeled tibiae with purified protocollagen hydroxylase suggested that a large part of the [ 14C]proline incorporated in 1 min was in polypeptides resembling collagen. Gel-filtration chromatography of extracts of pulse-labeled cartilage indicated that after short incubation periods with [ 14C]proline, significant amounts of [ 14C]proline were incorporated into molecules which were of about the same size as the complete polypeptide chains of α-collagen, but which did not contain significant amounts of [ 14C]hydroxyproline. Analysis of the elution patterns of 14C made it possible to estimate the time required to synthesize complete polypeptides de novo by a technique which apparently has not been used previously. The synthesis time calculated with this technique was about 1 min. Since maximal hydroxylation of collagen polypeptides in the system required over 10 min, the results indicated that cells synthesizing collagen under relatively normal conditions contain a significant intracellular pool of complete polypeptide precursors of collagen which are incompletely hydroxylated. A few proline residues may be hydroxylated while polypeptides are still attached to ribosomes, but the results suggested that most of the synthesis of collagen hydroxyproline occurs after completed polypeptides are released from ribosomal complexes.

The source of synovial fluid alkaline phosphatase.

SummaryAgar-gel electrophoresis shows that synovia! fluid alkaline phosphatase migrates differently from that of serum but similar to the enzyme of fresh cartilage and bone extracts. It is concluded that the syn-ovial fluid enzyme is derived from the articulating cartilage and not from serum. Purification of the synovial fluid enzyme by a method involving chromatography on TEAE-cellu-lose leads to modification of the enzyme that is accompanied by a change in electrophoretic mobility but not in its rate of sedimentation in a sucrose gradient. Changes in mobility of phosphatases occurred as the result of aging cartilage extracts or treating synovial fluid with neuraminidase. Chymotrypsin and trypsin, which do not alter the mobility, inactivate the phosphatase of cartilage.We thank Dr. Ben Moffett and Miss Janice Ruffing for performing the histological studies.

Acceleration of wound healing with heterologous cartilage. Immunological considerations.

SummaryA bovine cartilage preparation which has demonstrated therapeutic efficacy in accelerating wound healing in experimental animals and humans has been investigated for its potential antigenicity of the immediate type. Rabbits injected with an emulsion of this material with complete Freund's adjuvant did show the presence of precipitating antibodies while rabbits with implanted subcutaneous pellets showed no such activity. The precipitating antibodies produced did not cross react with human cartilage nor with other bovine tissues. Four groups of human subjects were tested intracutaneously with an aqueous extract of bovine cartilage. One laboratory worker with a history of clinical allergy and voluminous inhalation exposure of the material over a period of 8 years developed skin sensitizing antibody activity. Mild activity was demonstrated by 2 subjects who had received the cartilage therapeutically by topical application. These 3 subjects had no symptoms that could be attributed to sensitivity reactio...