Loretta D. Lee

Vertebral epidermal transamidases

Abstract The α-helical fibrous proteins (keratin) of the stratum corneum undergo changes in their physicochemical properties which are not solely explained on the basis of disulfide bond formation. The precursor of the stratum corneum protein, prekeratin, and the final product keratin were studied in the hair-follicle-free skin of calf snout epidermis. All of the lysines of prekeratin can be cyanoethylated while keratin molecules has 7–8 nmoles lysine per mg protein not available to cyanoethylation. Keratin from the human stratum corneum has 8–9 nmoles/g, protein which cannot be cyanoethylated. Direct examination of these proteins failed to detect the presence of N-e-acetyllysine groups or e-(γ-glutamyl)lysine groups as the explanation of the blocked lysines. The hair-follicle-free epidermis of several vertebrate species contains transamidases similar to the transglutaminases which form e-(γ-glutamyl)lysine crosslinks, in fibrin, hair follicle, and seminal fluid. The epidermal transamidase has a molecular weight between 40 000 and 60 000, is stimulated by reducing agents and calcium, and has most of its activity in the highermost levels of the epidermis. A semipurified preparation of the epidermal transamidase was able to form γ dimers and α polymers from Factor XIII-free fibrin. A transamidase substrate, [1,4-3H2]putrescine, when injected into mouse skin and studied autoradiographically, was localized in the stratum corneum. [1,4-14C2]putrescine incorporation into epidermal proteins was inhibited by 50 mM iodoacetamide. The incorporation of [1,4-14C2]putrescine was highest in very insoluble protein fractions. The exact role of transamidases in epidermal metabolism is not yet identified.

Purification of human intrinsic factor by affinity chromatography.

Abstract Hydroxocobalamin was insolubilized by covalent coupling to albumin which in turn was coupled to bromoacetyl-activated cellulose. This product allowed specific adsorption of cobalamin-binding proteins from human gastric juice. The cobalamin-binding proteins were eluted by increase of temperature and addition of cyanocobalamin. After further chromatographic purification the net result of pure intrinsic factor was about 5% or some 75 mg from about 500 1 of gastric juice. The biological activity was proven by the Schilling test. It was homogeneous by sodium dodecyl sulphate gel electrophoresis and immunoelectrophoresis. Stokes radius (3.3 nm) was the same as the one of native intrinsic factor in gastric juice. The partial specific volume determined by ultracentrifugation in water and in deuterated solvent was 0.743 cm 3 /g. The amino acid content was similar to the one of hog intrinsic factor. Sedimentation equilibrium ultracentrifugation indicated the presence of one or two protomers with a mol.wt of 30 000, unable to bind cyanocobalamin, and a monomer of mol.wt 60 000 binding one molecule of vitamin B 12 .

Matrix proteins of human hair as a tool for identification of individuals

A rapid, simple, reproducible and relatively inexpensive electrophoretic technique for displaying the structural proteins of hair is described. Hairs from a large number of individuals were examined and a number of distinct differences are presented. The possibility of using these individual differences as a means of individual identification is discussed.

The structural proteins of scaleless-mutant chick epidermis

Abstract Epidermis isolated from the anterior tarsometatarsus region of scaleless mutant chick legs was found to contain only α fibrous protein rather than the usual feather one. The polyacrylamide disc electrophoretic pattern of structural proteins isolated from mutant epidermis was also different from that of normal tissue. X-ray diffraction analysis of the claw of mutant chicks, however, showed the usual feather pattern. These results indicated that failure of scale induction rather than a defect in synthesis of feather protein is the abnormality in the mutant chick.

Chemistry and Composition of the Keratins

Keratinized tissues (epidermis, hair, feather, nail, claw, beak and horn) exhibit considerable heterogeneity in composition but they all contain structural proteins, designated keratins, as major constituents. In mammals these structural proteins include fibrous and nonfibrous proteins in varying proportions. A more generalized definition of keratins might include all products which result from the cornification of ectodermal cells, such as modified cell membranes and intercellular cementing substances. The processes involved in converting proteins in living, growing epithelial tissue into lifeless, tough, insoluble, translucent fibrous substances is termed keratinization .

Purification of thymidine phosphorylase from human amniochorion.

Thymidine phosphorylase (thymidine : orthophosphate deoxyribosyltransferase, EC 2.4.2.4) has been purified 1500-fold from extracts of human amniochorion. The purified enzyme catalyzes the phosphorolysis of deoxythymidine and to a lesser extent deoxyuridine but not deoxycytidine nor uridine. Discontinuous gel electrophoresis of the freshly purified enzyme shows a band containing 95% of the stainable protein. Gradient gel electrophoresis resolves the preparations into an active fraction with an apparent molecular weight of about 120 000 and a heavier less active or inactive fraction of about 180 000. Storage of the enzyme results in a decrease of the 120 000 dalton component, a loss in activity, and an apparent increase in the high molecular weight component. Sodium dodecyl sulfate gel electrophoresis shows only a single subunit of about 58 000 daltons which does not change on storage. These data are consistent with an active enzyme dimeric in structure which is capable of being converted to a less active form larger in molecular weight and possibly trimeric or tetrameric in structure.

Rocket immunoelectrophoresis in the presence of denaturing agents

Modifications of the Laurel rocket technique for assaying antigen antibody reactions are described. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents (e.g., SDS and urea) and subjected to electroimmunoassay without loss of sensitivity or specificity. Methods are also presented for obtaining rockets using gel slices from polyacrylamide gel electrophoresis either in the presence of urea or SDS. Results obtained using keratins, the insoluble proteins of epidermis, hair and nail are summarized.

The use of denaturing conditions for gel diffusion of insoluble epidermal proteins

Modifications of the classical technique for double diffusion in agar plates are presented. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents and antibody production specificity to be monitored by the appearance of precipitin lines in agarose gels as in the conventional Ouchterlony method. Results obtained using the insoluble proteins of bovine epidermis are summarized.

A neutral soluble high molecular weight keratin from epidermis

Abstract Previously described keratins, the structural proteins of epidermis, are soluble at pH values of less than 2.5, or greater than 10. At intermediate pH values, denaturants such as urea or SDS must be added in order to dissolve them, and in addition, with some types of keratin, disulfide reducing agents are also required to effect solution. These proteins are composed of several polypeptide chains in the molecular weight range of 45,000 to 70,000 daltons. This report describes the isolation and partial characterization of a new form of keratin, called neutral soluble keratin, which is freely soluble in low ionic strength buffers at neutral pH. The molecular weights of the polypeptides comprising neutral soluble keratin are in the range of 79,000 to 90,000 daltons.