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The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (>90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human a2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell ayer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collaren to localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form o collagen has an unusual affinity for the cell layer in this system.The class of proteins collectively referred to as collagen represents various distinct gene products. The synthesis of these proteins is somewhat tissue specific and is altered in pathological conditions (1, 2). At present, at least nine genetically distinct chains have been described as the primary constituents of various collagen molecules. Four collagen chains-the al(I), a2(I), al(II), and al(III) chains-comprise the interstitial collagens (1). Recent evidence indicates that five additional unique collagen chains are present in collagen molecules in basement membrane-like structures and in basement membranes. These are the aA and aB chains (3-6) and aC chain (7) isolated from highly vascularized tissue, and two additional chains obtained from lens capsule (8-10) and human placenta (11-13). However, little is currently known about the mechanisms that control the differential expression of the collagen genes. This paucity of information reflects, at least in part, the limited number of systems available for studying the regulation and dissecting the genetics of mammalian procollagen biosynthesis.This (5, 16). The origin of the CHL cell line as well as the clone (HT1) used in these studies has been described (14,17).Growth of Cells in Culture and Metabolic Labeling. CHL cells were maintained and grown in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum as described (14,15). For immunological studies, cells were grown in 15 X 60 mm dishes at 370C under a 10% C02/90% air atmosphere to t90% confluency. The medium was removed, the cell layer was washed with three 5-ml portions of cold phosphate-buffered saline, and the cells...