The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.

The susceptibility of three different solubilized forms of type IV collagen to gelatinase A cleavage and the concomitant effects on cell and integrin binding have been assessed. Dithiothreitol-solubilized Engelbreth-Holm Swarm (EHS) type IV collagen with disrupted intramolecular disulfide bonds in the CB3[IV] region was cleaved N-terminally to the CB3[IV] region into the two characteristic 100-300-nm fragments at 30 degrees C and was totally degraded at 37 degrees C. This was reflected in the partial or total loss of the alpha1beta1 and alpha2beta1 integrin binding sites within this region. The ability of gelatinase A to cleave EHS type IV collagen preparations with intact interchain disulfide bonds in CB3[IV] only occurred at higher temperatures. Furthermore, no effect on binding of cells or isolated integrins to the gelatinase-treated collagen could be detected after treatment at 37 degrees C. Dimeric collagen IV of human placenta with intact disulfide bonds in the CB3[IV] region was not degraded at all by gelatinase A at 37 degrees C.

[Changes of urinary hydroxylysylpyridinoline in patients with rheumatic diseases and after operation of prostheses of the hip joint.]. / Zmeny mocového hydroxylysylpyridinolinu u nemocných revmatickými chorobami a po operaci náhrady kycelního kloubu.

Urinary hydroxylysylpyridinoline (MHP), an indicator of the catabolism of cartilage and bone was assessed in patients with various rheumatic diseases - rheumatoid arthritis (RA) and ankylosing spopndylitis (AS). It was also assessed in patients with coxarthrosis (OA) and AS who had an endopro-thesis of the hip joint and in patients with OA who had a re-operation of a loosened total endoprothesis of the hip joint. A group of healthy subjects served as controls. In all groups of patients the MHP values were elevated as compared with controls. In operated patients different values were recorded - in patients with OA the values did not change after operation, while in AS a marked rise was found. Conversely, after re-operations a drop of MHP levels was observed. Key words: hydroxylysylpyridinoline, urinary excretion, rheumatic diseases, endoprothesis of the hip.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. V. Alternative method for preparation of serum albumin-methotrexate derivative.

An alternative method for the preparation of human serum albumin-methotrexate derivative (HSA-MTX) using N-hydroxysuccinimide ester of methotrexate (MTX) was compared with that using the water-soluble carbodiimide (WSC) assistance. The prepared derivative was tested to find the advantages and drawbacks of this method. The N-hydroxysuccinimide ester method is more laborious than that using WSC but some reasons speak in favor of this method. The formation of protein-protein conjugates during the reaction was negligible and under specific conditions more MTX molecules could be bound to protein than by the WSC method. However, some disadvantages of this method were found: A laborious preparation, a small degree of denaturation of protein during the synthesis and, moreover, the binding of MTX to protein did not always take place through NH2-groups of protein but through some other groups (--OH, --SH) as well. These couplings were not so stable as an amidic (or peptidic) bond. In water environment they were hydrolyzed and MTX was slowly released. If there is no difference in a small fraction of protein-protein conjugates, the WSC-assisted method possesses evident advantages over the active MTX ester one, mainly because of the simple preparation of the stable derivative.

Determination of radiolabelled proline and hydroxyproline in collagen hydrolysates by high-performance liquid chromatography with on-line radiometric detection.

Radiolabelled proline and hydroxyproline were separated on a C8 column (10 cm X 4.6 mm I.D.) with 10.4 mM sodium dodecyl sulphate in water-n-propanol (88:12, v/v) (pH 2.6) as the mobile phase at a flow-rate of 0.6 ml/min. The retention times of hydroxyproline and proline were 5 and 8 min, respectively. On-line radiometric detection was performed either in a homogeneous mode (liquid scintillator was added to the column effluent in the ratio 3.33:1) or in a heterogeneous mode (the detection cell was packed with a solid scintillator and 0.1 M ammonia was mixed with the column effluent in the ratio 1:6 in order to prevent adsorption of amino acids on the cell packing). Detection limits were in the range 100-900 dpm for individual isotopes and detection modes and the reproducibilities were better than 10%. The application of the method to a collagen synthesis study is reported.

Separation of type IX collagen from other cartilage collagens by hydrophobic interaction chromatography.

Collagen type IX was separated from other cartilage collagens (types II and XI) by hydrophobic interaction chromatography on a 25 cm X 8 mm I.D. stainless-steel column packed with Separon HEMA 1000 Bio. The mobile phase was 0.84 M ammonium sulphate with 0.1 M potassium dihydrogenphosphate (pH 6.5). Under these conditions only collagen type IX was eluted from the column; it could be monitored with UV detection (218 nm) or selectively with fluorescence detection (excitation 330 nm, emission filter 389 nm). The method can be used for the isolation and quantitation of collagen type IX. The assay was linear in the range 0-10 micrograms, the correlation coefficient was 0.99, precision 5.5% and accuracy 13%. The detection limit was about 0.6 microgram.