Biglycan (PG-I, BGN) and decorin (PG-II, DCN) are small proteoglycans that have been isolated in cartilage, skin, and bone. Although the function of biglycan is unknown, there is biochemical evidence that deeorin interacts with fibrillar collagens (type I, type II). The purpose of this study was to perform immunofluorescence and immunoelectron microscopy and immunoblotting of human embryonic and adult skin with antibodies directed against biglycan and decorin. These antibodies were developed against synthetic peptides of the core proteins of biglycan (amino acid sequence 11-24) and decorin (amino acid sequence 5-17). Immunofluorescence microscopy showed that decorin stained embryonic and adult collagen fib&. Biglycan did not stain collagen, but it appeared to stain the pericellular matrix of embryonic mesenchymal cells. Immunoelectron microscopy revealed labeling of all collagen fibrils with decorin antibodies regardless of their diameter, often at 60-nm perk&city. Positive stains suggest that most of the labeling was in the gap of the D-period (d and e bands) and also in one of the steps (c band). Decorin was identified by immunoblotting in fetal and adult skin. Also, significant amounts of core protein was identified lacking the dermatan sulfate chain. This study suggests that the core protein of decorin interacts with collagen fibrils although its specific function remains unknown. 0 1991 Academic Press, 1~.

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The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system. Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures. Sample meshes were processed for electron microscopy or immuno-electron microscopy. Fibrils approximately 20-30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate. As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter. By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen. No collagen fibril formation occurred in control cultures to which no ascorbate was added. However, large amounts of microfibrils were observed. Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35-54 nm in diameter. We believe that this system, which morphologically resembles a normal dermis, will serve as an excellent model for the study of collagen fibrillogenesis.

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S U M M A R YPolyphosphate kinase, an enzyme which incorporated the y-phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from Arthrobacter atrocyaneus by ammonium sulphate fractionation, DEAEcellulose column chromatography and Sephadex G-200 gel filtration. The enzyme had a broad pH optimum at 6.0 to 7-0 and required Mn2+ or Mg2+, histone, and inorganic phosphate for activity. The K , for Mn-ATP was 0.53 mM, and for inorganic phosphate was I -67 mM.Free ATP concentrations greater than 8 p~ inhibited the enzyme. Free Mn2+ or Mg2+ concentrations greater than 2 mM or 6 mM, respectively, were also inhibitory. Activity was strongly inhibited by 4 mM-ADP, I mM-PP, or 20 mM-NaF. The effect of ADP might have resulted from reversing the equilibrium of the kinase reaction. The activation by phosphate ions might indicate a role for the enzyme in regulating intracellular phosphate levels or maintaining a phosphorus reserve. The level of enzymic activity in the bacteria responded to changes in inorganic phosphate concentration in the medium. Basic proteins, such as protamine, could substitute for histone as activator. Proteins such as casein or bovine serum albumin would also substitute for histone but only in the absence of inorganic phosphate. The presence of a protein might be necessary to form a complex with the product, thus preventing reversal of the reaction in vitro.The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at IOO "C. It behaved as a long-chain-polyphosphate molecule on chromatography in an Ebel's solvent. The enzymic activity was therefore not that of a protein kinase.