Biochimica et Biophysica Acta (BBA) - Mucoproteins and Mucopolysaccharides最新文献

• 1.

  1. A spectrophotometric titration procedure for the estimation of chondroitin-sulphate using buffered toluidine blue has been developed. The titration readings are decreased by the action of hyaluronidase on chondroitinsulphate. Using this principle hyaluronidase has been assayed in amounts of 0.025–0.25 USP units. This range of activities corresponds with the depolymerisation of chondroitinsulphate at the rate of 45–450, μμequiv. glucuronic acid per min per 0.1 ml test solution.

• 2.

  2. The standard curves obtained with three different preparations of chondroitinsulphate differed little.

• 3.

  3. The assay is performed in the presence of 0.33 mM mercuric chloride. Ferric and ferrous salts at the same concentration inhibit the hyaluronidase activity. Crude serum albumin inhibits the activity slightly at the concentration present in blood.

• 4.

  4. The procedure has been shown to be applicable to the assay of hyaluronidase activity in fractions from blood serum and in serum itself.

• 5.

  5. The optimum pH of testicular hyaluronidase in the procedure and the nature of the products of the enzymic reaction have been studied.

The non-ultrafilterable material from pooled human urine was submitted to block electrophoresis and two fractions relatively rich in fucose but poor in other sugars and in proteins were further fractionated by various means. The material proved to contain a considerable number of fucose-rich glycopeptides, some of which were obtained in a reasonably pure state. The molecular weight was determined in three instances, giving values in the range of 5 000–10 000. Two of the glycopeptides were chemically characterized. One of them had a chemical composition very close to that given for purified blood group antigens. It was serologically inactive.

Treatment of washed human erythrocytes with the proteolytic enzyme pronase rapidly releases bound sialic acid from the membrane. The amount of bound sialic acid liberated is equal to the amount of free sialic acid which is released from this cell by neuraminidase (EC 3.2.1.18).

The bound sialic acid, liberated by pronase, was shown to be present in glycopeptides possessing M and N blood-group activity towards rabbit and human antisera. Using an MN active glycopeptide fraction, purified on Sephadex G-25, it has been possible by using DEAE-Sephadex to separate for the first time the M antigen from the N antigen. These glycopeptides which were obtained in a high degree of purity were shown to possess different carbohydrate compositions.

Vitamin A alcohol activates arylsulfatase (aryl-sulfatesulfohydrolase, EC 3-1.6.1) and inhibits β-glucuronidase (β-d-glucuronide glucuronohydrolase, EC 3.2.1.31) of rat colon. These effects were less pronounced with vitamin A acetate and they were negligible with vitamin A acid and other chemically related compounds. Action of vitamin A alcohol was not dependent upon the integrity of a particulate membrane and was unaffected by removal of lipid from the enzyme suspension, suggesting a direct effect on the enzyme proteins. Kinetics of arylsulfatase and β-glucuronidase were altered in the presence of vitamin A alcohol. Solubilized arylsulfatase and β-glucuronidase of particulate fractions from rat small intestine, kidney and liver showed the same response to vitamin A as the colon enzymes.

• 1.

  1. Incorporation of [I-¹⁴C]glucosamine into acid mucopolysaccharides was studied in connective tissue formed in stainless steel wire mesh cylinders implanted subcutaneously into adult rats. Liver, serum, and the fluids found within cylinders were also investigated.

• 2.

  2. High levels of non-dialyzable radioactivity were observed in tissue when labelled glucosamine was injected directly into the area of connective tissue proliferation within cylinders.

• 3.

  3. Incorporation of [I-¹⁴C]glucosamine into connective tissue was not adversely affected by removal of the animal's liver; only a small amount of radioactivity was found in serum proteins of these animals.

• 4.

  4. Incorporation of glucosamine by connective tissue slices was demonstrated by studies in vitro.

• 5.

  5. Mucopolysaccharides with high specific radioactivity, prepared from connective tissue labelled in vivo or in vitro, had staining characteristics and electrophoretic mobility of hyaluronic acid and chondroitin sulfate. Glucosamine and galactosamine, isolated from connective tissue, were found to be radioactive. It may be concluded that glucosamine can be directly incorporated into mucopolysaccharides at the site of connective tissue formation.

Ovine submaxillary glycoprotein (OSM) was digested exhaustively with pronase and the sialoglycopeptides separated by gel filtration. By subsequent neuraminidase (EC 3.2.1.18) treatment and further purification, glycopeptides of average molecular weight 660 and containing 50% of the total galactosamine of OSM were obtained. Serine and threonine were the only amino acids with a functional group in the side chain present in a concentration high enough to accommodate all hexosamine residues. Treatment of the glycopeptides with 0.5 N NaOH at 0° or 22° resulted in the release of N-acetylgalactosamine coincident with the loss of an equimolecular amount of hydroxyamino acids. Most likely the carbohydrate was released by the mechanism of β-carbonyl elimination. Some evidence for the formation of an unsaturated compound, presumably α-aminoacrylic acid, was afforded spectrophotometrically.

It is concluded that in OSM at least 50% of the prosthetic groups are joined glycosidically to the hydroxyl groups of serine and threonine.

When native OSM was treated at pH 8.0 and 42° for 120 h, about one-third of the prosthetic groups was released. After this treatment the isolated, non-dialyzable residual glycoprotein reacted strongly positive in the Warren test. The mechanism of the reaction is discussed.