Labelling of prolyl hydroxylase tetrameric subunits in freshly isolated chick-embryo tendon cells and in certain chick-embryo tissues in vivo.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) may be formed in the back reaction of the amino acid-activation reaction [Zamecnik, Stephenson, Janeway & Randerath (1966) Biochem. Biophys. Res. Commun. 24, 91-98]. On the basis of a number of observations of the properties of Ap4A it has been suggested that it may have a signal function for the initiation of DNA replication in eukaryotic cells] Grummt (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 371-375]. In the present paper human platelets have been shown to contain relatively large amounts of Ap4A. The compound is apparently metabolic inactive in platelets, but it is almost quantitatively released when platelets are activated to aggregate by treatment with thrombin. The results are discussed in connection with the known growth-stimulating activity of platelets.     

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.     

Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.     

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.     

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAs^Gly and iso-tRNAs^Pro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.     

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAsGly and iso-tRNAsPro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.     

Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen.

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.     

Uptake and 25-hydroxylation of vitamin D3 by isolated rat liver cells

The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.     

Decreased synthesis and increased intracellular degradation of newly synthesized collagen in freshly isolated chick tendon cells incubated with monensin

The synthesis and secretion of procollagen in embryonic chick tendon fibroblasts in suspension culture were inhibited with the carboxylic ionophore monensin. The synthesis of procollagen was inhibited by 50% in a 2-h exposure to 0.1 microM monensin and was inhibited by 70% in a 6-h exposure to 0.1 microM monensin. Secretion of procollagen was inhibited by greater than 90% in the 0.1 microM monensin-treated cultures and was totally inhibited by higher doses of the reagent. A cellular pool of collagenase-digestible peptides was demonstrated in the control cells, the level of which was elevated 3-4 times in the monensin-treated cultures. In order to determine whether the secretory and synthesis block caused by monensin inhibited intracellular degradation of newly synthesized collagen, the hydroxy[14C]proline in degraded collagen fragments present in control and monensin-treated cultures was determined and compared to the total hydroxy[14C]proline synthesized in each culture. The intracellular degradation of newly synthesized, pulse-labeled collagen was shown to proceed at rates comparable to those seen in the control cultures. The monensin-treated cells degraded pulse-labeled newly synthesized collagen nearly twice as long as the controls, resulting in an overall increase in the fraction of newly synthesized collagen that was degraded. These findings suggest that force generation in the activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations.     

DNA triple-helix formation on nucleosome core particles. Effect of length of the oligopurine tract.

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.     

Effect of proline on wound vessel member formation

Pretreatment of Coleus blumei (BENTH.) stem explants for 24hr with L-proline (P) (1.0 mM) by the successive cup techniqueresulted in the redifferentiation of extremely high numbersof wound vessel members (WVM) from pith and cortical parenchymatissues following the addition of a xylogenic concentrationof 5 ppm indoleacetic acid (IAA). The addition of P with auxinor after auxin had been introduced into the segments was ineffectivein altering the numbers of WVM formed as compared to controls.P-treated segments showed both normal and aberrant WVM withthe addition of colchicine (0.01 %) and IAA; colchicine-treatedsegments invariably produced aberrant WVM when P and IAA weregiven. These results suggest that a P-rich substance may interactwith auxin in initiating wound vessel member differentiation. ¹Fulbright Professor of Botany, 1967-68. Permanent address:Department of Biological Sciences, University of Idaho, Moscow,Idaho, 83843, U. S. A.     

Cortisol decreases the concentration of translatable type-I procollagen mRNA species in the developing chick-embryo calvaria.

The calvarial mRNA species of chick embryos were translated in the rabbit reticulocyte-lysate cell-free translation system. The amount of procollagen type-I mRNA species was determined by digestion with bacterial collagenase and by fluorography of the cell-free translation products. Administration of cortisol resulted in a specific decrease in the cellular concentration of translatable procollagen type-I mRNA species in the calvaria of developing chick embryos. There was a lag period of up to 12 h before the response, which was dose-dependent. The data suggest that the decrease in amounts of procollagen mRNA species is the main reason for the lower amount of tissue collagen after topical or systemic administration of glucocorticoids, although other factors may contribute to the response.     

Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells.

Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells were studied by using continuous labelling and pulse-chase labelling experiments with [14C]lysine. Control experiments with [14C]proline indicated that in continuous labelling the hydroxylation of [14C]proline became linear with time after about 4 min and the secretion of collagen after about 35 min, as reported previously. In similar experiments with [14C]lysine the hydroxylation of [14C]lysine and the glycosylations of hydroxy[14C]lysine became linear at about 4 min, suggesting that these reactions were initiated while the polypeptide chains were growing on the ribosomes. Pulse-chase labelling experiments with [14C]lysine indicated that after a 5 min pulse-label the hydroxylation of [14C]lysine and the glycosylations of hydroxyl[14C]lysine continued during the chase period for about 20 min. The data suggest that these reactions are continued after the release of complete polypeptide chains into the cisternae of the endoplasmic reticulum, whereas the reactions are probably not continued after the formation of the triple helix and the movement of the molecules into the Golgi vacuoles.     

Cell-to-cell signaling in the regulation of procollagen expression in primary avian tendon cells

Summary High cell density is required for high procollagen expression (50% of total protein synthesis) in primary avian tendon (PAT) cells but the signaling mechanism that triggers this response has been difficult to decipher. By using a quantitative in situ hybridization assay for procollagen mRNA, cell density dependent changes in procollagen expression can be followed at the single cell level. PAT cells can then be shown to respond to the presence of their neighbors over ∼1-mm distance. The cell density signal remains effective independent of the medium volume to cell ratio but becomes sensitive to dispersion and dilution when the medium is agitated. PAT cells respond to a reduction in cell density, when neighboring cells are scraped away, by outgrwoth (∼1 mm) and reestablishment of a cell density gradient in cellular procollagen mRNA levels. However, removing neighboring cells while preventing migration off of their own extracellular matrix retards the drop in procollagen mRNA levels. The evidence, taken as a whole, is consistent with a model whereby the cell density signal is a loosely bound component of the cell layer thereby restricting its diffusion to two dimensions but making it susceptible to dispersion by medium agitation. This work was supported in part by grant CA 37958 from the National Institutes of Health, Bethesda, MD, and in part by the Office of Health and Environmental Research, U.S. Dept. of Energy, Washington, DC, under contract DE-AC03-76SF00098.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Sources of reducing equivalents for the microsomal steroid 21-hydroxylation in isolated rat adrenal cells.

21-Hydroxylation of 11β-hydroxyprogesterone in the intact adrenal cells was stimulated by both glucose and pyruvate. Arsenite inhibited the basal as well as the pyruvatesupported reaction and also prevented the entry of pyruvate carbon into the Krebs cycle. Glucose-supported 21-hydroxylation was not inhibited by arsenite. It is proposed that NADPH for the microsomal 21-hydroxylation is derived by at least two mechanisms: (1) metabolism of glucose via the pentose shunt and (2) a mechanism involving transfer of reducing equivalents from the mitochondria into the cytosol. The latter would involve the transfer of some Krebs cycle intermediate (or intermediates) from the mitochondria to the cytosol followed by its eventual metabolism in the cytosol via an NADPH-linked dehydrogenase. This mechanism may assume importance when the cell has a limited supply of glucose.     

Effect of glutamine on fructose 2,6-bisphosphate and on glucose metabolism in HeLa cells and in chick-embryo fibroblasts.

Glutamine caused a dose-dependent decrease in fructose 2,6-bisphosphate concentration in both HeLa cells and chick-embryo fibroblasts. The effect was complete within 15 min in HeLa cells, but required more than 9 h in the fibroblasts. Half-maximal effects were obtained with 0.1-0.3 mM-glutamine. In chick-embryo fibroblasts, but not in HeLa cells, glutamine induced a time-dependent decrease in the activity of phosphofructokinase-2, which correlated with the decrease in fructose 2,6-bisphosphate. Glutamine decreased the glycolytic flux by about 25% only in chick-embryo fibroblasts. The difference in glycolytic response between the two types of cells might correspond to a difference in the sensitivity of phosphofructokinase-1 for fructose 2,6-bisphosphate. In HeLa cells, glutamine caused a 2-3-fold stimulation of the synthesis of glycogen, a 50% decrease in the concentration of fructose 1,6-bisphosphate and a more than 80% decrease in the concentration of 5-phosphoribosyl pyrophosphate; the concentrations of hexose 6-phosphates and ATP were not affected.