Frog Epidermis Research Articles

A new spectrophotometric method for the determination of cresolase activity of epidermis tyrosinase

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional frog epidermis enzyme, tyrosinase (E.C. 1.14.18.1), has been developed. It is a spectrophotometric method using p-coumaric acid and caffeic acid as substrates at pH 7.0. Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentrations. It is simpler, faster, and more economical than radiometric methods and, furthermore, permits continuous monitoring.

Activation of frog epidermis tyrosinase by immobilization on a solid support

1. Frog epidermis protyrosinase was activated by immobilization on Enzacryl AA and CNBractivated Sepharose. 2. The proenzyme immobilized through its tyrosine and lysine residues reached 28% and 19% of soluble enzyme activity, respectively. 3. Fluorescence spectra ( ι em), half-inactivation times (t 1 2 ), pH-activity profile and apparent Michaelis constant ( K′ m ) of immobilized protein were compared with that of the soluble enzyme. 4. The data obtained supported our previous hypothesis that activation mechanism of proenzyme involves a more unfolded conformational state.

Potassium reaccumulation by isolated frog epidermis

The involvement of potassium in transepithelial sodium transport was tested by studying net potassium reuptake by potassium-depleted frog skin epidermis. Normal potassium content in half-strength Ringer's (0.244 μequiv/mg dry weight) fell 43% after 16 h in K-free medium at 5°C. Reaccumulation, against an electrochemical potential gradient, to 83% of the initial tissue potassium content occurred following incubation for 4 h at 22°C in K-containing medium. Sodium was required in the solution bathing the inside, but not the outside surface of the skin, for net potassium reaccumulation. Ouabain caused an additional potassium loss from potassium-depleted epidermis, but did not have the same effect on potassium-depleted isolatedcells. Procaine, lithium and caffeine completely inhibited, antidiuretic hormone and cyclic AMP may partially inhibit and amiloride had no effect on potassium reaccumulation. In many cases decreases in sodium and water content were found to occur even in the absence of net potassium reaccumulation. The results suggest (1) potassium is actively transported into the epidermis, (2) this transport is not rigidly coupled to sodium extrusion or water loss, (3) potassium uptake is not rigidly coupled to transepithelial sodium transport, or only a small fraction is involved, (4) potassium diffusion is restricted in the extracellular space.

Stability of immobilized frog epidermis tyrosinase

Purified frog epidermis tyrosinase was immobilized on the following supports: CNBr-Sepharose 4B, Enzacryl AA, Enzacryl AH, Enzacryl Polythiolactone, and Enzacryl Polyacetal. The enzyme was active on all supports except when Enzacryl Polyacetal was used. The stability increased on immobilization. Enzacryl AA was the best support assayed. The Enzacryl AA-enzyme complex was 30- to 40-fold more stable to inactivation reaction than soluble enzyme, and maintained its activity when stored and assayed repeatedly. The immobilized enzyme on the other supports was also more stable than the soluble form. The pH-activity profile, thermal stability, storage stability, and the effect of protein concentration on activity of the immobilized enzyme have been studied. The properties observed for the immobilized enzyme were different than those of the soluble enzyme. The main reason for this difference could be due to enzyme modification through tyrosine groups of the enzyme; to conformational changes produced in the union to the matrix; and to microenvironmental differences created by the matrix.

Affinity chromatography of frog epidermis dopa-oxidase

Benzoic acid is a competitive inhibitor of the enzyme dopa-oxidase. p-Aminobenzoic acid was coupled by a single-step reaction of the amino group to: (1) CNBr-activated Sepharose 4B: (2) Enzacryl AA, by reaction with thiophosgene (pH 10.0); and (3) CM-Sephadex G-50, modified to azide by the Curtis procedure. These three solid supports were used as affinity adsorbents in the dopa-oxidase purification. The enzyme was obtained from frog epidermis, and was retained at pH 4.7 with 0.1 M acetate buffer. The enzyme elution was carried out using a linear pH gradient with 0.1 M phosphate buffer pH 8.0. We have examined the interaction of the enzyme with the immobilized PABA in relation to the nature of the support, the lengthening of the “arm” of the ligand, bathwise adsorption, and the enzyme activation by immobilized trypsin.

Adult amphibian epidermal proteins: biochemical characterization and developmental appearance

The keratin-like proteins (KLPs) from the epidermis of adult frogs of the species Xenopus laevis have been isolated and biochemically characterized by means of polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, amino-terminal end-group analysis and isoelectric focusing. One particular protein fraction of rather unusual amino acid composition found only in epidermal tissue was isolated in quantity by preparative gel electrophoresis and monospecific antibodies prepared against it. Using this anti-KLP antibody preparation it was possible to show that at least one kine of keratin-like protein characteristic of the adult epidermis first appears within the larval epidermis during metamorphosis. This is the first reported biochemical characterization of a tissue-specific protien from adult amphibian skin.

Some biochemical properties of Na,K-ATPase in frog epidermis

1. 1. Na,K-ATPase of frog epidermis was sensitive to proteases and easily destroyed. In contrast, Mg-ATPase was highly resistant towards proteolytic action. 2. 2. The Arrhenius plot of Na,K-ATPase activity shows a sharp discontinuity near 23°C; however, Mg-ATPase has no such critical point. 3. 3. Short-circuit current and Na,K-ATPase activity of the skin from a cold-acclimatized animal was not significantly different from that observed in a moderate-temperature-acclimatized animal. Mg-ATPase activity was lost around 50 per cent in cold-acclimatized animals; however, oxygen consumption of the skin was not significantly changed. 4. 4. The preparation obtained by Nakao's NaI method showed a high substrate specificity for ATP. The activity with CTP was about 10 per cent of that with ATP as substrate. Almost no ADP was utilized. The K m of the enzyme preparation for substrate ATP was calculated as 1·5×10 −3 M.

Flask cells and epidermal dynamics in frog skin

Variations in the fine structure of the flask cells of frog epidermis are described. Some flask cells have apical ridges with an alveolar layer of material under the cell membrane; others may have adjacent apical ridges sealed together by fusion of the outer leaflets of the cell membrane. The apical membrane of the flask cells and of the second tier epithelial cells (replacement or first reactive cells) is visibly different from the membranes on the lateral and basal aspects. Other variations in the appearance of flask cells may depend on their individual age, or on the stage of the moult cycle. The events of moulting can be divided into preparation, separation, sloughing and differentiation phases. During the preparation phase the flask cell apices disintegrate, and they are covered over by the adjacent replacement cells, which will later keratinize during the separation phase. New tight junctions are formed in the outer layers of the epidermis. Desmosome release frees the slough during the separation phase, before it is actually shed. During the differentiation phase the flask cells develop new apical ridges. The fine structure of the flask cells is compared with that of the mitochondria rich cells of the bladder epithelium, and with mitochondria rich cells which have been found in the palatal epithelium; the flask cells are considered not to be homologous with these other types. There are no comparable cells in the corneal epithelium. Possible functions of the flask cells are considered; some previous hypotheses can now be discounted. An attempt is made to integrate the phases of the normal moult with the variations in active sodium transport and water flux reported from isolated skin treated with aldosterone. It is suggested that the flask cells may transmit a hormonal stimulus causing release of the slough, but that they are not concerned in the effects on sodium transport, which can be explained by changes of the epithelial cells. The flask cells could however be involved in the transport of substances other than sodium.

A Proteolytically Activated Tyrosinase from Frog Epidermis

Abstract Tyrosinase (o-diphenol:O2-oxidoreductase; EC 1.10.3.1) is present in the epidermis of Rana pipiens pipiens as an inactive proenzyme that can be activated by a variety of endopeptidases, chymotrypsin being the most effective. This proenzyme has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. Inactive and active tyrosinase both have molecular weights of 200,000 ± 12,000 estimated by gel filtration over Sepharose 6B. The proenzyme contains 0.15 % copper. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates polypeptide molecular weights of 54,100 ± 700 for the inactive and 50,000 ± 700 for the active proteins. A peptide of molecular weight approximately 5,700 ± 1,100 appears on chymotryptic activation of the proenzyme. This peptide may be located on the NH2-terminal portion of the proenzyme since slow activation of the proenzyme occurs in the presence of leucine aminopeptidase. A tetrameric structure is suggested for both active and inactive enzymes. Tyrosine and 3,4-dihydroxyphenylalanine (DOPA) are oxidized with approximately equal Km of 5 x 10-3 m and kcat of 800 min-1. The enzyme is inhibited competitively with respect to DOPA by a variety of substituted benzoic acids. Inhibition by copper-complexing reagents such as diethyldithiocarbamate and cyanide can be reversed by addition of copper and there is evidence that copper may be reversibly removed from the active enzyme. Isoelectric points of 9.35 and 9.25 for the inactive and active enzymes, respectively, are consistent with the amino acid composition of the former material. A proteolytically activated tyrosinase from a related subspecies of frog Rana pipiens berlandieri exhibits an isoelectric point near six suggesting that the basicity of the enzyme is not related to its function. The utility of this enzyme-proenzyme system is discussed as an example of post-translational control of active enzyme levels.

High salinity adaptation of ion transport and epidermal lipids of frog skin

1. 1. Epidermal lipids and ion transport were measured in individual isolated frog skins and correlated. 2. 2. Significant lysolecithin-Na conductance and phosphatidylethanolamineCl conductance correlations were detected in the epidermis of unconditioned frogs. 3. 3. The absence of these lipid-transport correlations and a relative increase in sphingomyelin, found to be associated with suppression in active Na transport and Cl conductance in high salinity conditioning, could be related to the NaCI transport adaptive process. 4. 4. A correlation was found between relative proportions of several phospholipid fractions and Na resistance in conditioned frogs, compatible with phospholipid stoichiometry of some structure or substance in the Na transport pathway.

Horny cell formation in the epidermis of Rana pipiens.

AbstractWell preserved transitional cells were found between differentiated cells and horny cells of the frog epidermis, thus facilitating the study of the sequential events involved in horny cell formation. Autolysosomes appear to play an important role in the formation of horny cells. These structures preferentially digest those cytoplasmic components which are not necessary constituents of the terminal horny cell. The release of the contents of the small mucous granules into the intercellular spaces is one of the initial events in horny cell formation. Filaments and large mucous granules seem to be resistant to the lytic digestion and contribute to the bulk of the horny cell. Loss of fluids through the plasma membrane and consolidation of the remaining constituents, results in a flattened horny cell. The appearance of a thickened membrane around the horny cell signifies the completion of the transformation process.

A highly ordered structure in the frog epidermis

Crystal-containing granules were observed in the cytoplasm of the mid-region epidermal cells of the frog. These granules were usually ovoid with an average diameter of 0.3–0.4 μ m and were limited by an 85 A thick triple-layered membrane. Two types of crystals were observed within the granule. One consisted of rods, 102 A in diameter, arranged in a hexagonal lattice; and the other was a series of 47 A thick leaflets separated by a 23 A space. Highly ordered structures of this nature have not been reported previously in frog epidermis. Correlation of these granules with ordered structures found in keratizing epithelia of other vertebrate classes is discussed.

Mitotic activity in dorsal epidermis of Rana pipiens

1. l. Statistically significant rhythms of mitotic division were observed in dorsal epidermis of frogs, Rana pipiens, which had been exposed to a 12 : 12 light: dark environment for 14 days, and sacrificed at 2-hr intervals in groups of eight to twelve over a period of 24 hr. 2. 2. Male animals appear to have a primaty period length of 22 hr in the summer and 18 hr in the winter. 3. 3. Female animals have a period length of 18 hr. 4. 4. An inversion of the light régime for 14 days was not long enough to cause synchronization to this new lighting régime. 5. 5. Parapinealectomy and blinding abolished the rhythm.

The location of silver in frog epidermis after treatment by Ranvier's method, and possible implication of the flask cells in transport.

Frog skin, bladder wall, and sciatic nerve were treated by Ranvier's silver nitrate method and subsequently fixed and sectioned for electron microscopy. In the epidermis of the skin, more silver is found deposited in the flask cells than elsewhere, especially as a sub-apical plaque in the neck of the flask, which appears after the skin has been flooded with silver nitrate for 5 minutes. Mitochondria rich cells in the bladder also accumulate more silver than the surrounding epithelial cells, but do not show such a distinct sub-apical plaque. In myelinated nerve fibres treated similarly, silver accumulates in the paranodal regions of the axon, and outside the axon at the node. It is suggested that silver may accumulate near a site of ion transport, due to structural specialisations not visible by standard electron microscope techniques, and that the flask cells may therefore be implicated in transport in the frog skin.

Diurnal fluctuation in dna content and DNA synthesis in the dorsal epidermis of Rana pipiens

1.1. DNA content and DNA synthesis (3H-thymidine uptake) were measured in dorsal skin epidermis of frogs—Rana pipiens sacrificed in groups of 12 over a period of 24 hr—exposed to a 12 : 12 light-dark lighting régime for 2 weeks before sacrifice.2.2. Diurnal fluctuations were observed in both DNA content and DNA synthesis of dorsal epidermis.3.3. The reproducibility of these rhythms suggests that they are true physiological phenomena with periodicities of nearly 24 hr.4.4. The results of light inversion experiments suggest the importance of the lighting régime in the regulation of these rhythms.

Fine structure of clear cells in frog epidermis

The fine structure of clear cells located within the frog epidermis was investigated. Characteristic features of clear cells were, the ‘flask-like’ shape, the sparse amount of filaments resulting in a clear cytoplasmic appearance, abundant mitochondria, numerous small vesicles, and a system of convoluted apical membranes in cells adjacent to horny cells. These features were suggestive of a secretory function for the clear cell. A comparison of clear cells with fully differentiated epidermal cells (functioning in protection) was made, in an attempt to discover how structural differences between the two cells were related to the basic functions of the cells.

Ultrastructure of ovine ruminal epithelium and localization of sodium in the tissue

The rumen is lined by a keratinizing epithelium across which are transported large amounts of sodium and short-chain fatty acids. Maculae occludentes, but apparently not zonulae occludentes, are seen in the epithelium. Actively transported sodium may diffuse across the stratum corneum by way of the maculae occludentes and be pumped into the intercellular space in the midlevel of the epithelium. After appropriate fixation precipitates of sodium pyroantimonate are noted in the intercellular space and along cell membranes in the mid-epithelium. A barrier to the back diffusion of solutes may be formed by the narrow tortuous intercellular space, the polysaccharide coating on cornified cells, and the maculae occludentes. Thus, active transport of sodium across ruminal epithelium appears to have a similar morphological basis to that mechanism postulated for transport across frog epidermis.

Biochemistry of melanin formation.

Biochemistry of melanin formation.

The migratory behavior of frog epidermis, with special reference to surface polarity and to factors determining direction of movement

The migratory behavior of frog epidermis, with special reference to surface polarity and to factors determining direction of movement