J. Peter Bentley

The hydroxyproline of elastin

Abstract The question of whether the hydroxyproline found in elastin represents contamination with collagen was studied by subjecting solubilized elastin (α- plus β-elastin) to prolonged collagenase digestion. No loss of hydroxyproline was seen, and it is concluded that hydroxyproline is indeed a constituent of elastin in which it occurs in a collagenase-resistant sequence. The origin of this elastin hydroxyproline was studied by comparing the rate of incorporation of [14C]proline and hydroxy-[14C]proline into hydroxyproline of dermal and aortic collagen and aortic elastin. As has been repeatedly demonstrated for collagen, it was found that proline is a far better precursor of elastin hydroxyproline than is hydroxyproline itself. Both proteins incorporated a small amount of labeled hydroxyproline directly.

The biosynthesis of mucopolysaccharides and collagen by the sex skin of estrogen-treated monkeys

Swelling of the sex-skin was induced in five immature female rhesus monkeys by the administration of estrogen. The mucopolysaccharide content of this skin was compared with that of unswollen chest skin from the same animals and with untreated controls. The major difference was a 5-fold increase in the hyaluronic acid content of the sex skin. The biosynthetic rates of hyaluronic acid, dermatan sulfate, collagen, and non-collagen protein were studied by measuring the rate of incorporation of radioactively labeled precursors into these substances either in vivo or in vitro. A differential effect of the hormone was seen in that much more [14C]glucose was incorporated into hyaluronic acid and dermatan sulfate by sex skin than by non-sex skin. The incorporation of [3H]proline into collagen was also greater in the sex skin than in the non-sex skin. The rate of transfer of label from extractable toinsoluble collagen was, however, unaffected. Relatively little differential effect was seen in the incorporation of [3H]proline into non-collagen protein.

Structural Glycoprotein, Fact or Artefact

It has been repeatedly claimed that structural glycoprotein or SGP is a major component of many connective tissues, particularly aorta. SGP is usually defined as material extractable from tissue with chaotropic agents such as urea and having an amino acid composition matching that of other, similar extracts. It has been claimed that SGP comprises 10-60% of the dry weight of aortic tissue. This paper describes a study in which aorta was exhaustively extracted with agents previously claimed to extract SGP. All the extracts yielded mixtures of proteins which, upon separation by polyacrylamide gel electrophoresis, were found to consist largely of actin, collagen, myosin and other known proteins. No significant quantity of any one unidentified protein was found which could have been SGP. This study casts grave doubt upon the existence of SGP as a major component of aorta.

In vivo incorporation of labeled amino acids during early stages of collagen biosynthesis.

Abstract The biosynthesis of collagen has been extensively studied in many tissues by following the rate of uptake of labeled amino acids. These studies have been reviewed recently ( Jackson and Bentley, 1960 ). The various fractions reported by different workers all appear to arise by dispersion of tissue collagen aggregates into the constituent tropocollagen molecules, having specific physico-chemical properties ( Gross, Highberger and Schmitt, 1954 ). The fraction extracted with O. 14M NaCl, however, appears to contain the most recently synthesized collagen ( Jackson and Bentley, 1960 ) and hence is the most important fraction to study in the early stages of collagen biosynthesis. In most studies involving the uptake of isotopically labeled amino acids into collagen a regular pattern was observed showing a rise to a peak of activity followed by a fall to a minimum level. The time of peak activity and the rates of rise and fall depended on the type of extraction procedure and the tissue used, but in general only one peak of radioactivity has been observed. In carrying out experiments in which much earlier, and more frequent time points were studied than had been previously reported, we have noted an unexpected variation in the pattern of amino acid incorporation, both in skin and in open wound granulation tissue. It is the purpose of this report to describe and offer a possible explanation for the double peak uptake curve observed.

Salicylate‐induced inhibition of collagen and mucopolysaccharide biosynthesis by a chick embryo cell‐free system

The ability of a chick embryo cell‐free system to synthesize collagen, mucopolysaccharide and non‐collagen protein in the presence of sodium salicylate was studied. Added creatine phosphate together with endogenous creatine kinase was the ATP generating system. The incorporation of labelled proline or labelled glucose into collagen or mucopolysaccharide respectively depended on ATP level in the cell‐free system used. Salicylate inhibited collagen and mucopolysaccharide synthesis to a greater extent than non‐collagen protein synthesis. The ability of the cell‐free system to hydroxylate labelled protocollagen was inhibited 50% by storage at −18°. Ferrous iron reversed this inhibition. Salicylate prevented the restoration of the enzyme activity by ferrous iron. Incorporation of radioactivity into hyaluronic acid, when labelled UDP‐glucuronic acid and UDP‐N‐acetylglucosamine were supplied, was inhibited 17% by salicylate. Under the same conditions 47% inhibition of incorporation into chondroitin sulphate was seen. This suggests that UDP‐N‐acetyl‐glucosamine‐UDP‐N‐acetylgalactosamine epimerase is inhibited by salicylate.

Relation of collagen synthesis to chondroitin sulfate synthesis in cartilage

Abstract The question of whether the formation of mucopolysaccharide and collagen are mutually dependent processes was studied in an in vitro system. Puppy epiphyseal cartilage was incubated in the presence of sodium salicylate or a , a ′-dipyridyl in an attempt to inhibit the biosynthesis of mucopolysaccharides or collagen, respectively. The incorporation of glucose- 14 C or glucosamine- 14 C into isolated chondroitin sulfate and the incorporation of proline-H 3 into collagen hydroxyproline and into noncollagen protein were used as measures of biosynthesis. Salicylate markedly reduced chondroitin sulfate synthesis, presumably by inhibition of the enzyme l -glutamine- d -fructose-6-phosphate amino transferase. Since it was not possible to overcome this inhibition by supplying glucosamine, it was concluded that other enzymes involved in cartilage chondroitin sulfate synthesis are also inhibited, as has been demonstrated for the gastric mucosal synthesis of mucoprotein. Salicylate did not affect the production of noncollagen protein but greatly reduced the incorporation of label into the 0.14 M NaCl extractable collagen fraction. The incorporation into the 0.45 M NaCl extractable and insoluble collagen fractions was relatively unaffected. It was shown that the transfer of activity from the 0.14 M NaCl to the 0.45 M NaCl fraction and then to the insoluble fraction was increased in the presence of salicylate, and this was felt to be indicative of accelerated aggregation of collagen molecules into a cross-linked fibrillar form. The inhibition of collagen synthesis by a , a ′-dipyridyl was initially accompanied by relatively little effect on the chondroitin sulfate, but at the later time points a large reduction of labeled glucose incorporation was seen. As with salicylate, the addition of dipyridyl did not affect noncollagen protein synthesis, suggesting that general inhibition of cell metabolism is not the reason for the effects seen. When chondroitin sulfate was isolated and fractionated by a procedure dependent upon molecular weight differences, it was found that the radioactivity was associated almost exclusively with the higher molecular weight material. It was concluded that some degree of interaction exists between the biosyntheses of collagen and chondroitin sulfate.

The uptake of [14C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Abstract Rabbit ear cartilage was incubated with [14C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.

Collagen synthesis and fibrogenesis in scurvy.

Abstract A study was made of the uptake of labeled amino acids into collagen extractable with dilute salt solution from skin of ascorbic acid-deficient guinea-pigs. Greatly reduced collagen synthesis was demonstrated. By the use of different concentrations of extraction media, the degree of cross linking i.e. aggregation of the newly formed collagen into fibrils was followed. It is suggested that this aggregation is unaffected by ascorbic acid deficiency. By incorporating different labels ( 14 C and 3 H) into the collagen at 48-h intervals, the breakdown of newly formed aggregates was studied. Increased lability of these aggregates does not seem to be a factor in scurvy.

A SENSITIVE AUTOMATED PROCEDURE FOR THE DETERMINATION OF GLUCURONIC ACID

A newly automated method for the detection of uronic acids, particularly glucuronic acid is described. Data showing the limitations of this method are also presented. The advantages of the method are: (1) greatly increased sensitivity compared to the carbazole methods and increased range; (2) lowered interference from other sugars, proteins and salts; (3) variability to allow small sample volumes and/or continuous flow; (4) a more stable color reagent solution.

Biosynthesis of aortic collagen and glycosaminoglycan following immunological injury

The effect of immunological injury upon the early in vivo changes in aortic connective tissue metabolism was studied. It was found that a single antigen (bovine serum albumin), when administered in multiple doses, activated collagen synthesis and increased the rate of lgycosaminoglycan synthesis in the arterial wall. The degree of stimulation of both connective tissue components was higher in animals receiving a higher dose of antigen.