The collagen fibrils of human or guinea pig dermis when exposed to the denaturing agents, urea or guanidine-HC1, dissociated into smaller, disparate subunits, probably aggregates of microfibrils. The process of dissociation demonstrates that the fibrils are assembled helically. Initially, diagonal clefts appear on the surface of the fibril. These clefts are surface manifestations of a spirally oriented, internal space. Continued exposure to these denaturants resulted in progressive dissociation of the fibril into helically oriented subunits. It is suggested that water-miscible compounds such as glycols or hydroxypropyl methacrylate, in addition to the urea-guanidinium class of denaturants used in this study, affect the observed fbrillar changes through the disruption of hydrogen bonds between the microfibrils making up the fibril. Such a mode of action may explain why freeze-fractured or '~inert embedded" collagen demonstrates helical organization while other, more conventional methods of tissue processing do not. Further support for the proposed mode of action of these dissociative agents was provided by the observation that mature collagen, in which extensive intra-and intermolecular covalent crosslinks are present, is more resistant to dissociation than newly formed collagen.

Bovine comeal endothelial cells can be easily grown in culture using conventional techniques. The cultured cells closely resemble the parent, native endothelium. In culture the endothelium synthesizes and deposits, in a polar fashion, a well organized basement membrane that contains molecules which are characteristic of all basement membranes. Membrane deposition continues for at least a year and, during that time, it begins to acquire the unique, ordered substructure characteristic of the native membrane.

Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.

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