Characterization and localization of bovine epidermal transglutaminase substrate.

The substrate was purified by DEAE-Sepharose CL-6B ion exchange chromatography, Pevikon block electrophoresis, and ACA-54 gel filtration chromatography. This modified purification procedure resulted in a 19% substrate yield. Amino acid analysis of the purified substrate was performed following treatment in SDS, alkylation in 4-vinyl pyridine, and hydrolysis with methane sulfonic acid. Results included significant levels of lysine and glutamic acid, low levels of cystine and methionine, and a high amount of an amino acid eluting in identical position with authentic citrulline. Studies of the sedimentation equilibrium of the substrate showed a monodispersed protein with a molecular weight of 36,780 +/- 350. Ultrastructural studies of insoluble high molecular weight aggregates of the substrate revealed globular, amorphous masses lacking fibrillar structure. Using Ouchterlony double diffusion techniques, the initial substrate (36,000 m.w. substrate) and the soluble crosslinked high molecular weight substrate were shown to be immunologically identical. Indirect immunofluorescence was performed on freeze-dried sections of bovine snout epidermis using substrate specific rabbit antiserum and fluorescein-conjugated goat anti-rabbit IgG. Fluorescence was seen throughout the cytoplasm and inner cell membrane of granular cells, but was limited to the inner membrane of stratum corneum cells. Epidermal transglutaminase substrate is an amorphous, globular, citrulline containing protein synthesized in the cytosol of keratinizing cells with ultimate localization in the cell membrane.

Epidermal transglutaminase. Identification and purification of a soluble substrate with studies of in vitro cross-linking.

A Mr = 36,000 substrate for epidermal transglutaminase was identified immunochemically in buffer extracts of bovine snout epidermis, using antiserum to isolated high molecular weight substrate proteins recovered after cross-linking by transglutaminase. The high molecular weight proteins were not present in soluble epidermal extracts in EDTA prior to cross-linking. The substrate was purified by DEAE-Sepharose CL-6B and ACA-34 gel filtration, where it demonstrated an apparent molecular weight of 36,000. In the presence of Ca2+ and transglutaminase, the purified protein was converted to high molecular weight polymers which, under conditions of high protein concentration, included a protein aggregate insoluble in urea, sodium dodecyl sulfate, or beta-mercaptoethanol. Cross-linking did not occur with Ca2+ alone or in the presence of EDTA or putrescine, a competitive inhibitor. The epsilon-(gamma-glutamyl) lysine isodipeptide was identified in high molecular weight products of cross-linking but not in the Mr = 36,000 precursor. We postulate the intracellular assembly of Mr = 36,000 substrate, with subsequent cross-linking by transglutaminase and insolubilization into the keratinocyte membrane.

New findings on epidermal transglutaminase substrates.

Recent studies suggest the presence of natural substrates for epidermal transglutaminase in buffer extracts of epidermis. We have investigated a soluble substrate of 36,000 molecular weight present in neutral buffered extracts of cow snout epidermis. When purified to homogeneity, this protein was cross-linked by transglutaminase with 70-80% efficiency into a series of high molecular weight soluble polymers and a highly insoluble protein aggregate. The epsilon-(gamma-glutamyl) lysine bond was present in both high molecular weight soluble polymers and insoluble aggregate, but was absent from the initial 36,000 molecular weight protein. Substrate cross-linking was completely inhibited by EDTA, iodoacetamide, and 25 mM putrescine. When subjected to electrophoresis in gels containing sodium dodecyl sulfate (SDS), 90% of the 36,000 molecular weight protein was reduced to subunits of 8,000-10,000 molecular weight. We postulate intracellular synthesis of the 36,000 molecular weight initial substrate molecule, followed by insolubilization and cross-linking at the cell membrane.

Control of amino acid transport in the mammary gland of the pregnant mouse.

The regulation of the uptake of the amino acid analog alpha-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulin-treated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein in subject to negative feedback regulation from the intracellular amino acid pool.