Assembly of chick and bovine lens-capsule collagen.

Chick-embryo and adult bovine lens-capsular epithelia in organ culture synthesized 4-hydroxy[3H]proline-containing polypeptides when incubated in the presence of [3H]proline. These collagenous polypeptides of apparent Mr 180 000, 175 000 and 160 000 became incorporated with time into aggregates of higher molecular size. The formation of such aggregates was inhibited when the tissues were labelled in the presence of beta-aminopropionitrile, thereby implicating lysine-derived cross-links in aggregate formation. When the tissues were incubated in the presence of tunicamycin, the collagenous polypeptides synthesized exhibited increased electrophoretic mobilities on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The addition to lens-capsule incubation medium of alpha alpha'-bipyridine led to the synthesis of underhydroxylated type IV collagen, also of increased electrophoretic mobility. Extended pulse-chase experiments indicated that such underhydroxylated collagen did not participate in aggregate formation, but was at least as stable as fully hydroxylated non-cross-linked collagen synthesized in the presence of beta-aminopropionitrile. Native type IV collagen, recovered from the culture medium when capsules were incubated with [3H]proline for 24h, was purified by ion-exchange chromatography. Separations conducted on CM-cellulose under denaturing and nondenaturing conditions suggested that the alpha 1(IV) and alpha 2(IV) chains occur in the same heterologous triple helix. Densitometric analyses of appropriate fluorograms indicated that these two polypeptides occur in a 2:1 ratio, suggesting that lens-capsule collagen is synthesized as a triple-helical molecule of composition [alpha 1(IV)]2 alpha 2(IV).     

Studies on the assembly of the rat lens capsule. Biosynthesis of a cross-linked collagenous component of high molecular weight.

1. Intact rat lenses in tissue culture synthesize hydroxy[3H]proline-containing polypeptides of apparent mol.wt. approx. 180000, which become assembled into aggregates of higher molecular weight with time. 2. Both the 180000-mol.wt. species and the aggregates are components of the deoxycholate-insoluble base-membrane matrix. 3. Formation of the high-molecular-weight aggregate is accompanied by the biosynthesis of the reducible hydroxylysine-derived cross-link hydroxylysino-5-oxo-norleucine. 4. Hydroxylysino-5-oxonorleucine and dehydrohydroxylysinonorleucine are the major reducible cross-links present in intact foetal and 1-month-old calf lens capsules.     

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.     

Glycoprotein synthesis in explants of developing rabbit mammary gland in culture.

1. Explants of mammary glands of pregnant rabbits cultured in the absence of insulin, prolactin and cortisol incorporated [2-3H]mannose into lipid-linked mono- and oligo-saccharide and protein. 2. Inclusion of the hormones in the culture medium stimulated the incorporation of [2-3H]mannose into lipid-linked monosaccharide 4-fold, into lipid-linked oligosaccharide 4-fold and into protein 13-fold after 24 h in culture. 3. Addition of tunicamycin to the incubation medium completely inhibited the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and protein after an initial lag period of about 2h. Incorporation of this radiolabel into lipid-linked monosaccharide was increased 4-fold under these conditions. 4. Incorporation of [4,5-3H]leucine into protein was unaffected by the presence of tunicamycin. 5. Analysis of mannose-labelled protein by polyacrylamide-gel electrophoresis indicated that a major radiolabelled protein of apparent mol.wt. 65,000-70,000 was synthesized and approx. 70% of this protein appeared in the soluble fraction. 6. Glycosylation of the protein but not synthesis of its peptide backbone was sensitive to tunicamycin. 7. Possible origins of this glycoprotein synthetized when the tissue is stimulated to differentiate in culture are discussed.     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.     

The effect of cycloheximide on the glycosylation of lactating-rabbit mammary glycoproteins.

1. Cycloheximide inhibited immediately the incorporation of L-[4,5-3H]leucine and D-]2-3H]mannose into mammary proteins, suggesting that the mannosylation of mammary glycoproteins requires the continued supply of newly synthesized polypeptides. 2. The incorporation of radioactivity from N-acetyl-D-[1-14C] glucosamine into protein was not inhibited until approx. 30 min after cycloheximide addition. Much (greater than 90%) of this radioactivity was present as N-acetylgalactosamine. 3. N-Glycosylation appears to be inhibited immediately by cycloheximide due to a lack of newly synthesized acceptor polypeptides, whereas O-glycosylation continues for 30 min, the time taken for acceptor peptides to move from their site of synthesis to the Golgi region and for completion of glycosylation. 4. There was a transient increase in the incorporation of mannose into lipid-linked oligosaccharide in the presence of cycloheximide, followed by a decrease in the radioactivity in this fraction. 5. The major lipid-linked oligosaccharide extracted from explants incubated for 2h in the presence of cycloheximide (6-7 monosaccharide units) was smaller than that extracted from control explants (10-12 monosaccharide units).     

Preferential usage of glycyl-tRNA isoaccepting species in collagen synthesis.

Chick embryo tRNA charged with [3H]glycine was incubated in an in vitro protein-synthesizing system using polysomes isolated from either chick embryo liver or calvaria. Using collagenase digestion to measure the fraction of protein synthesized which was collagenous, the results indicate that in the calvaria system approximately 65% of the incorporated [3H]glycine was in collagen. The incorporation of [3H]glycine into protein from individual isoaccepting species was determined by chromatography on a reversed phase system of the charged tRNA before and after incubation in the polysome systems. In the calvaria system, a single tRNAGly species cognate to GGU and GGC and which is found in unusually large amounts in collagen-synthesizing tissues was used preferentially in collagen-synthesizing tissues was used preferentially in collagen synthesis. In the liver system, the rate of incorporation was similar to the calvaria, but no collagen synthesis was detected and only a relatively small preferential usage of any of the four major isoaccepting species was observed. These results support the notion that the complement of tRNA found in a cell may be adapted to the synthesis of a particular protein. It is also possible that under certain circumstances, collagen synthesis may be controlled in vivo at the translational level by the concentration of particular tRNA species.     

Programmed synthesis of collagen during postembryonic development of the nematode Panagrellus silusiae.

The relative rate of collagen synthesis in the free-living nematode Panagrellus silusiae during postembryonic development was found to be discontinuous by measuring either the incorporation of tritium into material extracted as collagen or the amount of collagen-bound tritiated proline and hydroxyproline after 2-hr incubations of whole worms with [³H]proline. A peak of collagen production preceded each of the three molts that were examined. Moreover, protocollagen prolyl hydroxylase activity during each intermolt period paralleled the pattern of collagen synthesis. On the other hand, a triphasic pattern was not observed when noncollagenous proteins were labeled with either [³H]tryptophan or [³H]leucine. In addition, the level of soluble radioactive proline that accumulates in whole organisms after 2-hr incubation periods did not fluctuate appreciably during postembryonic development. The mean ratio of hydroxy-proline to proline in a number of collagen samples extracted at various times during the maturation phase was 0.113 ± 0.040. Pulse and chase experiments with [³H]proline indicated that most of the collagen synthesized during a peak period is lost after the second ecdysis following the labeling interval. In contrast, a considerable proportion of the collagen synthesized during nonpeak periods is retained throughout the postembryonic period. It is postulated that the modulated pattern of collagen biosynthesis in Panagrellus reflects, for the most part, a quantitative regulation of the production of cuticular collagen during postembryonic development.     

Elastin biosynthesis in chick-embryo arteries. Studies on the intracellular site of synthesis of tropoelastin.

Electrophoretic analyses of the products of cell-free translation of elastin mRNA isolated from 17-day chick-embryo thoracic arteries have demonstrated that the elastin mRNA codes for polypeptides that are slightly larger than the cellular tropoelastin polypeptides synthesized and secreted by matrix-free artery cells. Pulse-chase experiments with cells labelled with [3H]proline established that newly synthesized tropoelastin polypeptides were associated solely with membrane-bound particulate fractions. Cell-free translation of membrane-bound and free polyribosomes isolated from artery cells revealed that the tropoelastin mRNA was associated predominantly with the membrane-bound fraction. When rough-microsomal fractions, isolated from cells labelled with [3H]proline for 10 min, were treated with proteinases in the presence and in the absence of detergent, the nascent tropoelastin polypeptides were shown to be susceptible to proteolysis only when the integrity of the membranes was destroyed by detergent treatment. In similar experiments tropoelastin polypeptides synthesized by membrane-bound polyribosomes in the nuclease-treated reticulocyte lysate were also resistant to the proteolytic-enzyme treatment. The results suggest that tropoelastin polypeptides are synthesized on membrane-bound polyribosomes and discharged into the lumen of the endoplasmic reticulum with co-translational removal of a signal peptide.     

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.     

Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide.

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as 'protein 21"). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as "protein 33"), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.     

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells.Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins.These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion.Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.     

Rate of basement membrane biosynthesis as an index to angiogenesis

A method was developed for assessing collagenous protein biosynthesis from [U-14C]proline in relation to angiogenesis in the chick chorioallantoic membrane (CAM). The rate of collagenous protein biosynthesis both in vitro and in vivo was maximum between days 8 and 11 of chick embryo development. This was the stage of maximum angiogenesis as shown by morphological evaluation of the vascular density. At day 10 the rate of collagenous protein biosynthesis was 11-fold higher than that of day 15, when angiogenesis had reached a plateau. The collagenous protein formed by CAM co-elutes on SDS-agarose chromatography with the collagenous component of [3H]-acetylated-basement membrane (BM) from bovine lens capsule. 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), which was shown previously to be a specific inhibitor of BM collagen biosynthesis, caused about 80% reduction in collagenous protein synthesis by CAM. These results indicate that most of the collagenous protein synthesized by CAM was BM collagen and this can be used as a biochemical index of angiogenesis.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.     

Translation of collagen messenger RNA in a cell-free system derived from wheat germ.

A cell-free system for synthesizing protein from wheat germ was used to translate the messenger RNA extracted from 16-day embryonic chick calvaria. A part of the product had properties similar to collagenous peptides and served as a substrate for prolyl hydroxylase, an enzyme specific for collagen. The level of potassium was critical for the synthesis of high molecular weight products with properties similar to pro-alpha-chains. The potassium concentration for optimal protein synthesis, as judged by maximum incorporation of [3H]proline into acid precipitable material, was considerably lower than the concentration required for the synthesis of high molecular weight collagenous peptides.     

Biochemical changes in rat kidney on exposure to elemental mercury vapor: effect on biosynthesis of metallothionein.

Evidence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a metallothionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rats which were injected with [U-14C] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-14C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporation of 109Cd into the fraction followed the same pattern as that of [14C] cystine in rats injected with tracer doses of CdCl2 labeled with radioactive 109Cd isotope. This 10 000 mol. wt. protein, newly synthesized in response to repeated exposures to mercury vapor, exhibited identical properties to metallothionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [U-14C]-cystine into this protein in rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney.     

The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO(2) (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [(3)H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO(2) [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [(3)H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO(2) [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO(2) for the first 48h were unable to respond to a subsequent increase in pO(2) during the last 24h. Analysis of pepsin-solubilized collagen alpha-chains labelled with [(14)C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO(2). Type-III collagen comprised 20-30% of the radioactivity in alpha-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO(2) was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo.     

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.