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A sensitive and specific radioimmunoassay was developed for the precursor-specific peptide segment located at the amino end of bovine type III procollagen. Human material showed high cross-reactivity in this assay. Two forms of human procollagen peptides were detected in body fluids. The larger peptide (45K) was found in serum and ascites, and resembled the whole precursor-specific segment which is presumably released from human type III procollagen by a single enzymatic cleavage. The smaller peptide (10K) was found mainly in urine indicating that further degradation of circulating procollagen peptides is required prior to their passage through the kidney. Compared to peptide concentrations in normal human serum two to twenty-fold increases were observed in all patients with alcoholic liver disease, in fifteen of seventeen patients with acute hepatitis, and in ten of fourteen patients with chronic active hepatitis. Much higher levels were detected in ascites fluid. Patients with rheumatoid arthritis and other diseases showed far smaller elevations of the serum peptide. In alcoholic liver disease peptide levels correlated well with inflammation and necrosis observed in liver biopsies, but not with other laboratory parameters.

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Laminin was recently characterized as being a major non-collageneous protein of the basement membrane matrix produced by a mouse tumor. It was extracted from the tumor matrix with neutral buffer and purified under non-denaturing conditions. Rabbit and guinea pig antisera raised against laminin or a large pepsin fragment P1 of laminin showed strong binding to both laminin and peptide PI but only a weak reaction with reduced and alkylated laminin. The major antigenic determinants of laminin were located in a disulfide knot, comprising one third of the molecule, which resisted degradation by pepsin or cyanogen bromide. Minor antigenic determinants shared by the native and reduced protein could also be identified. The data were interpreted as showing that laminin consists of conformationally rigid as well as more flexible domains. Absorption studies with mouse kidney homogenate indicated that authentic basement membranes contain a protein immunologically identical to laminin. Tissues from other species contain a related protein which exhibits partial cross-reaction with mouse laminin. Together with immunofluorescence data the findings demonstrate that laminin, like type IV collagen, occurs in most basement membranes of the body.Basement membranes are composed of special forms of collagen (type IV) and of other glycoproteins which are still incompletely characterized [l 1. Tumor models such as the mouse EHS sarcoma [2] and a teratocarcinoma cell line [3] have been recently used to prepare basement membrane proteins in amounts sufficient for comprehensive chemical and immunological studies. A large non-collageneous protein which we have named laminin [4,5] could be extracted by neutral buffers from the EHS sarcoma and purified to electrophoretic homogeneity. This protein consists of at least two disulfide-bonded polypeptide chains ( M , 220000 and 440000) and was found to differ in several chemical and immunological properties from fibronectin, another major glycoprotein of connective tissue [6,7]. Immunofluorescence analyses [4,5] have suggested that a protein, identical or related to laminin, also exists in authentic basement membranes. In the present study we describe immunochemical properties of laminin and provide further evidence that it is an ubiquitous protein of basement membranes in normal tissues. MATERIALS AND METHODS Purijication of LamininThe mouse EHS sarcoma was propagated subcutaneously in the C57B1 strain [2] and harvested when the tumor reached a weight of 5 -15 g. Laminin was extracted from the tumor with 0.5 M NaCl, 0.05 M Tris-HCl pH 7.4 and purified by chromatographic methods. The purified material showed essentially a single band when examined by sodium dodecylsulfate electrophoresis [4]. Chemical and Proteolytic Degradation of LamininDisulfide bonds of laminin were reduced with 0.02 M dithioerythritol in 8 M urea and were subsequently S-carboxymethylated or S-aminoethylated [8]. Sodium dodecylsulfate electrophoresis of reduced and alkylated laminin showed two bands with apparent molecular weig...

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Murine neuroblastoma cells have been widely used as a model system for neuronal cells as they can be induced to differentiate in culture by various stimuli, such as dibutyryl cyclic AMP (dbcAMP), prostaglandin, and serum starvation. The cells respond with assembly of microtubules, leading to neurite outgrowth, with increased activity of neuronal-specific enzymes, tyrosine hydroxylase, choline acetyltransferase and acetylcholine-esterase, and synthesis of neurotransmitters. The differentiated cells lose tumorigenicity. Cell-to-substratum adhesion is evidently crucial for neurone extension in vitro. Neurite outgrowth is induced by treatments that increase cell-to-substratum adhesion in some neuronal cell cultures. We have now identified the major high molecular weight proteins synthesized and secreted by murine C1300 neuroblastoma cells as fibronectin, laminin and type IV procollagen, of which the latter two were also found to be deposited in pericellular matrix form.