Changes in sugar nucleotide and high energy phosphate kinetics during in vivo development of sponge biopsy connective tissue. G G Bole J Clin Invest. 1966;45(11):1790-1800. https://doi.org/10.1172/JCI105483. Research Article Find the latest version: https://jci.me/105483/pdf Journal of Clinical Investigation Vol. 45, No. 11, 1966 Changes in Sugar Nucleotide and High Energy Phosphate Kinetics during In Vivo Development of Sponge Biopsy Connective Tissue * GILES G. BOLE t WITH THE TECHNICAL ASSISTANCE OF JANET C. LEUTZ (From the Rackham Arthritis Research Unit, Department of Internal Medicine, The University of Michigan, Ann Arbor, Mich.) Connective tissue varies widely in its histo- shown to be essential to the anabolic synthesis logical appearance and chemical composition. Its of proteins (7), polysaccharides (8), and lipids physical state ranges from the hardness of bone (9). The monosaccharide sugar nucleotides are to the viscous mucopolysaccharides found in syn- the precursors presumed to be involved in the ovial fluid. Current evidence would indicate that biosynthesis of mucopolysaccharides, glycopro- the special fibrillar and other extracellular con- teins, and glycolipids (8, 10, 11). In several ex- stituents are formed by the cells of the connec- perimental systems (12) these intermediates have tive tissue (1). Certain of these constituents been shown to be involved in formation of the have been shown to change with age (2, 3), nu- products of connective tissue cells, and partial tritional status (4, 5), and during experimental characterization of nucleotides present in car- propagation in vivo (6). Observations of this rageenin granulomas has been made by Decker type suggest that physiological factors that regu- and Gross (13). late the functional state of connective tissue may In the current study, the 5'-ribonucleotides pres- be directly associated with the chemical inter- ent in acid soluble extracts of 14-, 28-, and 42- mediates leading to de novo synthesis of these day sponge biopsy connective tissue have been cell products. The 5'-ribonucleotides 1 have been isolated and identified. This time interval spans the most active period of connective tissue or- * Submitted for publication April 1, 1966; accepted ganization of subcutaneously implanted polyvinyl August 12, 1966. This study was supported by grant AM 06206 from sponge in experimental animals (14). The con- the National Institutes of Health, U. S. Public Health tent of UDP-N-acetylhexosamines was found to Service. The Rackham Arthritis Research Unit is sup- increase with tissue age, whereas the concentra- ported by a grant from the Horace H. Rackham School of tion of UDP-hexoses and GDP-hexoses decreased Graduate Studies. A preliminary report of this work has been published (Arthr. and Rheum. 1963, 6, 793). between 14 and 42 days. No significant shift in t Senior Investigator, Arthritis Foundation. the per cent composition of the other 5'-ribonu- Address requests for reprints to Dr. Giles G. Bole, cleotides was demonstrated. The incorporation The Rackham Arthritis Research Unit, The University of 32P-orthophosphate into purine and pyrimi- of Michigan, University Hospital, Ann Arbor, Mich. dine nucleotides was also studied at each tissue 1 The following abbreviations are used: AMP, ADP, age. ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP for the 5'-mono-, di-, and triphosphates of Methods adenosine, cytidine, guanosine, and uridine; UDP-G for Production of inflammatory connective tissue in guinea uridine diphosphate glucose; UDP-Gal for uridine di- pigs. The technique involved in implantation and removal phosphate galactose; UDP-hexose for unresolved mix- of polyvinyl sponge granulomas and the morphologic tures of UDP-G and UDP-Gal; UDP-AcGm for uridine characteristics of the tissue have been previously de- diphosphate-N-acetyl-D-glucosamine; UDP-AcGalm for scribed (14). Four sponge implants (0.5 X 2 X 2 cm) uridine diphosphate-N-acetyl-D-galactosamine; UDP- were made in the low dorsolumbar area of adult mongrel AcHm or UDP-N-acetyl-hexosamines for an unresolved guinea pigs (650 to 800 g); 15 to 2Q animals were used mixture of UDP-AcGm and UDP-AcGalm; UDP-GA for UDP-D-glucuronic acid; GDP-M for guanosine di- GDP-Fu, and GDP-X; Pi for inorganic phosphate; Pt phosphate mannose; GDP-Fu for guanosine diphosphate for total phosphate. In initial fractionation A260 = ab- fucose; GDP-X for a guanosine diphosphate monosac- sorbance of ultraviolet (UV) light at 260 mgu in a 1-cm charide; GDP-hexose for the total amount of GDP-M, light path X total volume in milliliters. 1790 SPONGE BIOPSY CONNECTIVE TISSUE 5'-RIBONUCLEOTIDES 1791 in each experiment. Two groups of animals were studied tilled water, and final purification and separation of the at 14 days, and one group was studied at 28 and at 42 individual nucleotides were accomplished by paper chro- days. Weight of the animals was determined every 3 matography. After application to Whatman 3-mm paper, days throughout the period of experimental observation, descending chromatography was carried out with iso- and individual animals were excluded from the study butyric acid: concentrated ammonium hydroxide: water unless weight stability or gain was recorded. Each ani- (57: 4: 39), pH 4.3. The ultraviolet-absorbing bands mal's drinking water was supplemented with ascorbic were identified, eluted with water, and concentrated, and acid for 5 days after implantation of the polyvinyl each one was rechromatographed with 95% ethanol: 1.0 sponges. Four hours before removal of the implants, M ammonium acetate (70: 30), pH 7.4. The ultraviolet- 5 Ac of Na2H32PO4 was injected into each of the absorbing bands were again eluted with distilled water; granulomas. these eluates were used to identify the individual nu- Preparation and fractionation of the acid soluble tissue cleotides. extract. The sponge implants were rapidly removed from Identification of 5'-ribonucleotides. The mobilities (Re) each animal, cut into small fragments, and placed di- of the nucleotides separated by paper chromatography rectly into an equal volume of cold (40 C) 10% tri- were compared with the mobility of standard 5'-ribonu- chloroacetic acid. Wet weight of the total tissue mass cleotides 3 chromatographed as marker compounds on the was determined, and all subsequent operations were car- same chromatogram. Total absorbance at 260 mu was ried out at 40 C. The implants were homogenized in a determined for each component, and ultraviolet absorp- Waring blendor for 5 minutes. The supernatant was tion spectra at pH 1.5, 5, and 11 were determined in a filtered through a coarse scintered glass funnel and the Cary model 14 recording spectrophotometer. The purine residue re-extracted with an equal volume of 5% tri- or pyrimidine base was identified by the characteristic chloroacetic acid. The total extract was combined and spectral shifts that occur at these pH values. After we the final acid soluble fraction obtained after repeating had previously determined total absorbance at 260 my the filtration. Trichloroacetic acid was removed by three for each component and identified the base present in each, successive extractions with equal volumes of diethyl we used the appropriate molar absorbancy index (21) to ether. The volume of water soluble material was mea- calculate the molar concentration of each nucleotide iso- sured and absorbance at 260 mu (A260) and 280 mku de- lated from the tissue. NAD was identified by its ab- termined in a Beckman DU spectrophotometer. Analyses sorption spectrum and the characteristic spectral shifts for inorganic phosphorus, total phosphorus (15), adenine in absorption maxima that occur at 260 and 327 my (16), and protein (17) were carried out on portions of after the addition of cyanide. Samples from the same this material. Preliminary fractionation of the extract preparations were analyzed for phosphorus by the Fiske- was accomplished by stepwise gradient elution from a Subbarow method as modified by Hurlbert, Schmitz, Dowex 1 (formate) x-8, 200- to 400-mesh resin column Brumm, and Potter (15). Susceptibility of these nucleo- 30 X 1.9 cm in size. The column was washed with 400 ml tides to enzymatic hydrolysis by 5'-nucleotidase present in distilled water followed by elution with increasing concen- Crotalus adamanteous venom was carried out according to trations of formic acid and ammonium formate in formic the method of Heppel and Hilmoe (22). To assay mono-, acid as described by Denamur, Fauconneau, and Guntz di-, and triphosphate nucleotides, we employed a 100- (18). Serial 15-ml fractions were collected on an auto- fold excess in enzyme concentration to simultaneously matic fraction collector, and the A260 of each fraction was utilize the pyrophosphatase as well as the 5'-nucleotidase determined. One-ml samples from each fraction tube were activity of the venom (11). One-hour incubations were removed for measurement of radioactivity. The pres- analyzed for inorganic and total phosphorus (15), and ence of Ninhydrin reactive material in the column frac- zero time incubations served as controls. tions before the addition of ammonium formate to the Characterization and identification of sugar nucleo- eluent was determined on 0.05-ml samples after they were tides. In this study 0.2-,umole portions of all uridine, applied to Whatman 1 paper (19). guanosine, and cytidine nucleotides having a mobility on Appropriate fractions were pooled, and the pH of the final preparative paper chromatography similar to the pooled eluates was adjusted to 4.5 with NH40H. This respective sugar nucleotide standards were hydrolyzed material was absorbed on 30- X 1.9-cm columns of coarse with 0.01 N HCl at 1000 for 15 minutes. The hydro- SGL acid-washed charcoal.2 The columns were washed chloric acid was removed by lyophilization and the hydro- with 500 to 1,000 ml of distilled water, and this was lysate analyzed chromatographically as shown in Figure promptly followed by the rapid elution of the charcoal 1.