The initial steps in the processing of the common precursor to adrenocorticotropin (ACTH) and endorphin in mouse pituitary tumor cells (AtT-20) have been investigated. Three forms of the precursor have been resolved by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with apparent molecular weights of 29 000 (29K ACTH-endorphin), 32 000 (32K ACTH-endorphin) and 34 000 (34K ACTH-endorphin). These forms have a similar peptide backbone, but their carbohydrate content differs. In particular, a tryptic glycopeptide has been observed in 32K ACTH-endorphin which is not present in 29K ACTH-endorphin and has been identified as the tryptic peptide containing the alpha(22--39) sequence of ACTH. Similar heterogeneity in carbohydrate has been observed in some of the smaller molecular weight forms of ACTH which are resolved by NaDodSO4 gel electrophoresis. Pulse chase and continuous labeling studies using radioactive amino acids and sugars suggest that the 29K ACTH-endorphin is converted to 32K and 34K ACTH-endorphin by the addition of carbohydrate. The glycopeptide and pulse chase studies suggest that 29K ACTH-endorphin is at a branch point in the processing pathways. It can either be converted to 4.5K ACTH by proteolytic processing or to 32K ACTH-endorphin by the further addition of carbohydrate. The 32K ACTH-endorphin can then be converted to 13K ACTH, the glycosylated form of 4.5K ACTH (Eipper, B.A.,& Mains,, R.E. (1977) J.Biol. Chem.252, 882), by proteolytic processing. A comparison of the distribution of the different molecular weight forms of ACTH and endorphin in mouse pituitary extracts and in the mouse pituitary tumor cells reveals that the pituitary contains all of the forms of ACTH and endorphin seen in the tumor cells, including the three forms of the ACTH-endorphin precursor. However, the molecular weight distribution of the forms in the anterior lobe is very different from that in the intermediate lobe of mouse pituitary.

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine y-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of -90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.During terminal differentiation, keratinocytes of the epidermis and other stratified squamous epithelia synthesize an envelope consisting of cross-linked protein beneath the plasma membrane (1). Localization of the envelope appears due to the presence of a membrane-bound transglutaminase (2, 3) and several of its substrate proteins at the cell periphery (4). The enzyme is anchored in the membrane by acylated fatty acid (5) and is activated by flux of Ca2+ into the cytoplasm when cellular membranes lose their integrity during the final maturation stage (6). The biochemical events resulting in mature envelopes have been difficult to follow due to the intractable nature of the highly cross-linked product. In view of the many proteins and amines in keratinocytes serving as transglutaminase substrates (4, 7), further study of the enzyme structure may help in analysis of this process. In addition to acylation, for example, phosphorylation of the membrane anchorage region has been seen, which could alter the interaction of the enzyme with potential substrate proteins (8).The blood clotting factor XIII catalytic subunit (9-11) and tissue transglutaminase (12) have recently been cloned and sequenced. These enzymes are distantly related to each other but display significant similarity in certain regions, especially around the active site. An origin of the latter region in common with thiol proteases has been proposed (9). The more recent demonstration of striking similarity between the active site and a corresponding region in the erythrocyte band-4.2 protein (13, 14), however, indicates that closer relatives of transglutaminases exist. A cDNA clone for the keratinocyte-specific enzyme of the rabbit was originally identified by using an oligonucleotide probe directed toward the active site and was partially sequenced (15). Using that clone as probe, we have now cloned and determined the complete primary structure of this third type of transglutaminase for the human and rat.O Although many type...

Alteration of gene expression by inorganic arsenic has been studied in cultured human keratinocytes derived from normal epidermis, a premalignant lesion and a malignant tumor. The purpose was to find whether these cells displayed common alterations in gene expression that might elucidate the mechanism of arsenic action. Global analysis of approximately 12 000 genes by microarray showed that approximately 30% were expressed. Of these, transcription of a substantial fraction (up to 12%) was altered, nearly twice as many being suppressed as stimulated by 2-fold or more at 2 micro M sodium arsenite or 6 micro M arsenate, which did not affect cell growth. At 0.67 micro M arsenite (50 p.p.b.), effects on transcription were less pronounced but clearly evident. Genes whose transcription was altered in common among all the treated keratinocytes included those induced by reactive oxygen, of which heme oxygenase-1 displayed the highest fold induction. Genes indicative of reactive oxygen generation were detected at the earliest time examined, raising the possibility this feature drives subsequent cellular responses. Unlike some agents that produced transient induction of heme oxygenase-1, arsenicals produced sustained induction. Comparison with other agents producing reactive oxygen in the cells, as reflected in heme oxygenase-1 induction, suggested cellular differentiation was suppressed by sustained but not transient generation of reactive oxygen. Sustained global changes in gene expression were seen in target cells treated chronically with inorganic arsenic at concentrations consumed by millions of humans in contaminated drinking water.

During the adipose conversion of 3T3 cells, there occur sequential changes in cellular protein and mRNA composition. To determine if there are also changes in transcription, we have studied the transcripts of specific genes in isolated nuclei prepared before and after adipose conversion. Transcription of three genes encoding adipocyte-specific proteins was detectable only in adipocytes, whereas transcription of actin and collagen type I genes occurred in both adipocytes and preadipocytes. The activation of transcription of adipocyte-specific genes was not synchronous. Thus temporal differences in the appearance of different adipocyte mRNAs probably result from differences in the times of activation of transcription.

Keratinocyte transglutaminase is anchored on the cytosolic side of the plasma membrane by fatty acid thioesterification near the amino terminus, a process which is seen to occur within 30 min of synthesis. The importance of a cluster of five cysteines (residues 47, 48, 50, 51, and 53) where acylation was presumed to occur is now demonstrated by site-directed mutagenesis. Transglutaminase mutants in which the cluster is deleted or the cysteines are all converted to alanine or serine are cytosolic. Partial replacement of the cluster, leaving two contiguous cysteines, is sufficient to confer membrane anchorage, while a single cysteine is only partially effective. As demonstrated with a soluble transglutaminase mutant, membrane anchorage confers susceptibility of the amino-terminal region to phorbol ester-stimulated phosphorylation. Attachment of 105 residues from the transglutaminase amino terminus to involucrin, a highly soluble protein, results in membrane anchorage of the hybrid protein. Attachment of the cysteine cluster alone does not result in membrane attachment of involucrin, but a 32-residue segment containing this cluster is sufficient. Stable transfectants of the human transglutaminase in mouse 3T3 cells are membrane-bound, indicating the fatty acid transacylation is not keratinocyte-specific.