Isolation of Hyaluronateprotein From Human Synovial Fluid*

Sandson, John; Hamerman, David

doi:10.1172/jci104639

Cited by 73 publications

(18 citation statements)

References 21 publications

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“…Zone electrophoresis of hyaluronidase-digested human PP-L was performed on blocks of polyvinylchloridet (Sandson and Hamerman, 1962) at pH 7.4 (0 04 M phosphate) at 15 volts/cm. for 6 hrs.…”

Section: Methodsmentioning

confidence: 99%

Immunofluorescent Studies of Human Articular Cartilage

1966

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The matrix of articular cartilage is produced by chondrocytes and is composed of chondromucoprotein (CMP) enmeshed in a network of coUlagen fibres. Native CMP consists of chains of chondroitin sulphate and keratin sulphate combined with protein to form large complexes. These complexes can be extracted from cartilage by highspeed homogenization in water (Malawista and Schubert, 1958). Ultracentrifugation of CMP from human articular cartilage at 105,000 G. yields two proteinpolysaccharide fractions; one sedimentable, PP-H, and one non-sedimentable, PP-L (Gerber, Franklin, and Schubert, 1960). PP-L is the purer of these fractions and in the adult is composed of approximately equal parts of protein, chondroitin sulphate, and keratin sulphate.Previous histological studies of the distribution of CMP in articular cartilage have depended upon the metachromatic or other anionic staining properties of chondroitin sulphate and keratin sulphate. In normal human articular cartilage there is diffuse metachromatic staining of the matrix with a slight diminution in the intensity of the staining in the most superficial zone adjacent to the joint space. In osteo-arthritis the superficial zone of articular cartilage becomes fibrillated and vacuolated, and exhibits a striking loss of metachromatic staining (Collins, 1949).In this paper we describe the use of an immunofluorescent technique to study the distribution of CMP in normal and osteo-arthritic articular cartilage. Specific antisera to CMP were prepared in rabbits and an indirect fluorescent antibody technique was used to localize the CMP in sections of cartilage. The immunofluorescent and metachromatic staining patterns of normal cartilage were *

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Section: Methodsmentioning

confidence: 99%

Immunofluorescent Studies of Human Articular Cartilage

1966

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“…The methods used to isolate hyaluronate are described in detail elsewhere (2). About 10 to 40 g of fluid from individual joints was diluted 1:4 with a buffer (0.04 M phosphate, 0.1 M NaCI, pH 7.2) and dialyzed at 50 C for 24 hours in 500 ml of this buffer changed twice daily.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, viscosity of the sol was much lower than the viscosity of that part of the same sample of HP which was prevented from forming a gel by prior trypsin digestion (Figure 3). Although the nature of the association of HP chains that leads to (2).…”

Section: Nature Of Isolated Hyaluronateproteinmentioning

confidence: 99%

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Unusual Properties of Hyaluronateprotein Isolated From Pathological Synovial Fluids*

Hamerman¹,

Sandson²

1963

J. Clin. Invest.

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In joints actively involved by rheumatoid arthritis, the nature of the synovial fluid is quite different from normal. The fluid is increased in volume (10 to 30 ml) and contains large numbers of leukocytes (> 10,000 per mm3) . The concentration of protein (4 to 5 g per 100 ml) is more than twice that of normal synovial fluid, whereas the concentration of hyaluronate (0.1 g per 100 ml) is reduced to one-third to one-half normal. Many studies have been made of the physical properties of rheumatoid synovial fluid (1), and although these reflect the physical state of the hyaluronate in the fluid, the hyaluronate has not been isolated for study.Recently, mild methods were described to isolate hyaluronate from normal synovial fluids (2). Hyaluronate containing about 2% protein was obtained, and this product was called hyaluronateprotein (HP). Similar methods were used to isolate hyaluronate from rheumatoid synovial fluids, and a number of unusual findings were observed. A compound of hyaluronate combined with more protein (about 10%o) was isolated from the majority of the fluids; some of these products formed gels during dialysis in acetate buffer at pH 4.5, and all showed immobilization at the origin during zone electrophoresis at pH 4.5. None of these properties of the hyaluronate in rheumatoid synovial fluids has been previously described. During the course of these studies, the question arose whether such findings were specific for HP from rheumatoid fluids. It appears that they are not, for in a preliminary study of fluids from a few

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“…It remains possible, as with the 2% of protein that Sandson and Hamerman (1962) showed to be firmly bound to the hyaluronic acid of human synovial fluid, that some polypeptide or protein is chemically bound to the hyaluronic acid in ox synovial fluid. However, in view of the finding of Silpananta, Dunstone, and Ogston (1968) that undegraded hyaluronic acid can be separated with as little as 0·5% of protein, this amount must be very small.…”

Section: Discussionmentioning

confidence: 99%

Protein Associated with Hyaluronic Acid in Ox Synovial Fluid

Silpananta¹,

Dunstone²,

Ogston³

1969

Aust. Jnl. Of Bio. Sci.

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SummaryProtein which is associated with hyaluronic acid, prepared in a physicochemically unchanged state from ox synovial fluid by ultrafiltration, has been separated by preparative equilibrium sedimentation in a caesium chloride density gradient and by chromatography on DEAE-Sephadex. Earlier work has been confirmed by showing that the protein consists of at least two chemically distinct lipoprotein, components. It has also been confirmed that its removal from the hyaluronic acid causes marked changes of viscosity, without the occurrence of degradation of the hyaluronic acid. The separated proteins appeared in a number of differently aggregated forms, and this fact has prevented our drawing conclusions about their molecular sizes in synovial fluid.

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Isolation of Hyaluronateprotein From Human Synovial Fluid*

Cited by 73 publications

References 21 publications

Immunofluorescent Studies of Human Articular Cartilage

Immunofluorescent Studies of Human Articular Cartilage

Unusual Properties of Hyaluronateprotein Isolated From Pathological Synovial Fluids*

Protein Associated with Hyaluronic Acid in Ox Synovial Fluid

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