Control of keratin (K) gene expression may be important for cell differentiation in complex epithelia such as salivary gland. To investigate differences in distribution between keratin mRNAs and their respective proteins, a combined in situ hybridization (ISH) and immunohistochemical study was undertaken on nine normal salivary glands and seven pleomorphic adenomas. ISH employed riboprobes to K7, K8, K14, K18, and K19. Immunohistochemistry was performed on adjacent sections using monoclonal antibodies (MAbs) to the same keratins. Normal luminal cells showed abundant hybridization with probes for K7, K8, K18, and K19. Keratin 14 mRNA was present in basal and myoepithelial cells at a low level of expression. Proteins of their keratins were strongly stained. Neoplastic cells showed variable expression of mRNA and protein for K7, K8, K18, and K19. There was a high level of K14 mRNA but variable protein. The findings provide evidence that expression of these keratins in normal salivary epithelia is regulated transcriptionally and that in neoplasia this system is in considerable disarray.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

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A method is described for the immobilization on tritylated agarose or Sepharose columns of a wide spectrum of enzymes, including types useful in contemporary biochemistry/molecular biology, many of which have never before been reported as immobilized. The method involves the formation of noncovalent hydrophobic bonds between the enzymes and trityl groups which are attached to the agarose by means of ether bonds. The immobilization of calf intestinal and E. coli alkaline phosphatases to tritylagarose is reported in detail. Their binding strength, binding capacity, and long-term stability (greater than six months) are described as a function of the salt concentration, pH, buffer type, and degree of agarose substitution. Homologies are noted between tritylagarose-bound and membrane-bound phosphatases. This method compares favorably with other methods, covalent or otherwise, reported to date, in terms of the enzyme immobilization yield (ca. 100%), the mildness of conditions, resulting, in most cases, in the retention of a high degree of activity, the ease and speed of the manipulations, and the long-term stability of the immobilized enzyme. Further, it is noted that highly tritylated and crosslinked Sephadex G10 selectively and mildly removes detergents from enzyme solutions.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a fulllength keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.