Prenylated proteins: the structure of the isoprenoid group.

The mevalonate-derived portion of a prenylated protein from Chinese hamster ovary cells has been established as diterpenoid (C20). This group is linked to a carboxyl-terminal cysteine as a thioether. It was removed from the protein by hydrazinolysis followed by Raney nickel desulfurization, and the resulting hydrocarbon fraction was analyzed by gas chromatography-mass spectrometry.

Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo.

Exposure of normal human skin in vivo to ultraviolet irradiation at wavelengths shorter than 320 nanometers stimtulated an unscheduled DNA synthesis in all of the cell layers of the epidermis and in the upper dermnial fibrocytes. The skin of patients with xeroderma pigmentosum did not show this response. correlation of these findings with previous tissue culture studies suggests that the defect in repair of the damaged DNA in xeroderma cells occurs in vivo as well as in vitro.

Role of sweat in accumulation of orally administered griseofulvin in skin.

Griseofulvin, an orally effective antimicrobial agent, appears in the stratum corneum within 4-8 h after oral administration. Griseofulvin distribution was found to be highest in the outermost layers of the stratum corneum (level I, 20.8+/-1.5 ng/mg) and lowest inside (level II, 10.0+/-1.5; level III, 7.5+/-2.2 ng/mg). In order to study the precise mechanism of griseofulvin transfer to stratum corneum, the role of sweat in the accumulation of griseofulvin was considered. Heat-induced total body sweating decreased the mean stratum corneum concentration of griseofulvin by 55%, and 200-300 ng of griseofulvin accumulated per ml of sweat. A silicone hydrophobic resin was used to differentiate between "wash-off" and carrier properties of sweat for griseofulvin. Prevention of transepidermal water and sweat loss by (a) topical application of formaldehyde-releasing cream to one palm, (b) occlusion by a 2 x 2-cm patch on one arm, and (c) wearing a rubber glove for 24 h, showed a lower griseofulvin concentration when compared to control areas in the same subjects. The results of the gloved hand experiment show that a complete equilibrium is established at all three levels of stratum corneum, thereby removing the reversed gradient. These results support the hypothesis that a "wick effect" is responsible for the observed reversed drug gradient within the stratum corneum. The results of the experiments suggest that sweat and transepidermal fluid loss play an important role in griseofulvin transfer in stratum corneum.

In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Antibody-dependent cell-mediated cytotoxicity in selected autoimmune diseases.

Antibody-dependent cell-mediated cytotoxicity mediated by peripheral blood lymphocytes was studied in patients with systemic lupus erythematosus, polyarteritis nodosa. Sjogren's syndrome, and rheumatoid arthritis. The target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody. Antibody-dependent cell-mediated cytotoxic activity was normal in Sjogren's syndrome and rheumatoid arthritis but significantly decreased (P is less than 0.001) in active systemic lupus erythematosus and in two patients with polyarteritis nodosa. A partial regeneration of antibody-dependent cell-mediated cytotoxic activity was obtained by treatment with pronase and DNase followed by overnight incubation. Sera from patients with systemic lupus erythematosus inhibited antibody-dependent cell-mediated cytotoxic activity of normal lymphocytes. The inhibitory activity was studied by specific immunoadsorption and sucrose density geadient ultracentrifugation. Removal of IgG but not IgM greatly reduced inhibition. Inhibitory factors were present in 7S and heavier fractions containing IgG. Five systemic lupus erythematosus patients were studied serially to determine if improvement in clinical status could be correlated with a decrease in serum inhibitory factors as studied by inhibition of normal antibody-dependent cell-mediated cytotoxicity. Indeed, a greater serum inhibitory capacity was found in each patient during periods of greater disease activity.

Discrimination between Rb+ and K+ by Escherichia coli.

1. The K+ requirment of Escherichia coli is only partially fulfilled by Rb+. The molar growth yield on Rb+ was about 5% of that on K+ and the growth rate in Rb+-supplemented media is lower thatn in K+ influx by any of the four K+ transport systems of E. coli. The high-affinity Kdp system (Km = 2 micron) is poorly traced by 86Rb+. It discriminates against a 86Rb+ tracer at least 1000-fold. The two moderate affinity systems, the high-rate TrkA system (Km = 1.5 mM) and the moderate rate TrkD system (Km = 0.5 mM), discriminate against a 86Rb+ tracer by approximately 10-fold and 25-fold, respectively. 86Rb+ is preferred by the low-rate TrkF system and overestimates its K+ influx by 40%.

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis.

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure

Purification and characterization of bradykinin-hydrolyzing enzyme from 2-day-old rat epidermis.

Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.

Purification and some properties of bound cysteine proteinase inhibitor from cornified cells of newborn rat epidermis.

Cysteine proteinase inhibitor exists in two forms in terminally differentiated keratinocytes. One is readily soluble in 20 mM sodium phosphate buffer but the other is bound to the plasma membrane and is poorly soluble. The cysteine proteinase inhibitor (CPI) from the membrane was extracted from cornified epidermal layers of 2-day-old rats and its properties were compared with those of soluble CPI. This CPI (bound CPI) was solubilized in alkaline 8 M urea containing 2-mercaptoethanol from the residual tissue exhaustively treated with buffered 4 M urea. CPI was separated from keratin by ammonium sulfate precipitation and purified by means of papain affinity chromatography, ion exchange column chromatography and gel filtration. Bound CPI had an Mr value of about 16,000, a pI value of 3.8 and was unstable at above 80 degrees C, while soluble CPI was of Mr 13,000 and stable at above 80 degrees C. Both CPIs were stable at 4 degrees C in the range of 3.0-9.0. Bound CPI contained half cystine and the ratio of acidic-to-basic amino acids was 3.18. Bound CPI inhibited rat liver cathepsins B, H, and L but did not inhibit the activity of noncysteine proteinases. Papain activity was inhibited by bound CPI at three sites, noncompetitively, and the Ki value was calculated to be 0.11 nM.

Immunochemical studies of proteins in epidermal cornified cells of human and newborn rat.

1. Proteins were extracted from cornified cells of newborn rats and human palm with 8 M urea containing 0.1 M beta-mercaptoethanol. Two fractions, rat FIIIa and human F5.5 were obtained by acid precipitation for further study. 2. Antibodies raised in rabbits by injection of rat FIIIa gave two precipitin lines by agarose diffusion against rat FIIIa, but only one line against human F5.5. One of the antigenic determinants of rat FIIIa was found to be a protein of approximately 66 000 daltons. The other seems to be formed with two polypeptides in the range of 60 000 and 66 000 daltons. The antigenic determinant of human F5.5 was a protein of approximately 64 000 daltons which immunologically cross-reacted only with the antiserum to a protein of 66 000 daltons in rat FIIIa. 3. The antisera also cross-reacted with proteins extracted from epidermis of guinea pig, hamster, hairless mouse, dog ear, dog snout and dog foot pad, but did not react with the epidermis of either rabbit immunized with rat FIIIa or non-treated normal rabbit. 4. Indirect immunofluorescence demonstrated a reaction of rabbit anti-rat FIIIa serum over cornified cells as well as over granular, spinous and basal cells of the epidermis of newborn rat and human.

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis.

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.

Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells.

(1) Combination of techniques for extraction and purification of histidine rich protein established by several investigators were employed for comparison of histidine-rich protein in granular cells and cornified cells of newborn rats. (2) Histidine-rich protein extracted from the same cell fraction by two different techniques either in 1 M potassium phosphate buffer (Ugel) or in 4 M urea (Dale) showed identical elution profiles on CM 52 cellulose ion exchange chromatography and the same SDS polyacrylamide gel electrophoretic patterns. (3) Histidine-rich protein from granular cells contained polypeptides of larger molecular sizes than those in histidine-rich protein from cornfield cells, although amino acid composition of the two histidine-rich protein was non-distinguishable (histidine residue was more than 7%). (4) Antibodies raised in rabbits by injection of histidine rich protein from granular cells and that from cornfield cells immunologically cross-reacted. Furthermore, the antisera were found to be reactive over both keratohyalin granules and cornified cells, but not epidermal cells of the lower strata.

Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli.

Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane. In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent. In the mutant lacking KdpB the other two subunits copurified under the same conditions. It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex. A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data.

Light-scattering studies of cation-stimulated filament assembly of newborn rat epidermal keratin.

Effects of CaCl2 on in vitro polymerization of keratin extracted from cornified cells of newborn rat were investigated by means of light-scattering and supramolecular structures. Elongation and parallel assembly of filaments occurred with addition of CaCl2 to dialyzed keratin solutions and was detected by an increase in light-scattering intensity. Nonfibrous aggregates which occurred in higher buffer concentrations and in lower pH were also recorded as intensity increased. MgCl2, ZnCl2, and GdCl3 demonstrated similar effects, but NaCl and KCl showed no effect.

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells.

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.

Cation transport in Escherichia coli. VI. K exchange.

K influx and net K flux have been measured in suspensions of chloramphenicol-arrested Escherichia coli. The rate of K exchange in the steady state was independent of the K concentration of the medium over a 200-fold range. Under a number of experimental conditions the rate of exchange may be considerably increased or decreased without changing the cellular K content. These results show that under these conditions changes in K influx are associated with equal changes in K efflux, and suggest that the latter process is, at least in part, both carrier-mediated and tightly coupled to the influx process.

Cation transport in Escherichia coli. IX. Regulation of K transport.

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.

Cation transport in Escherichia coli. VIII. Potassium transport mutants.

Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.

Cation transport in Escherichia coli. VII. Potassium requirement for phosphate uptake.

When Escherichia coli K-12 is grown in media containing limiting amounts of K, growth continues normally until all the extracellular K has been consumed. Thereafter the rates of growth, glucose consumption, and oxygen consumption decrease progressively, and the cell contents of K and P fall. These changes, referred to as K limitation, are all reversed by the addition of K. By specifically altering the ionic composition of the cells it was shown that these metabolic disturbances are not due to changes in the cell content of K or Na, but are directly related to the absence of K from the extracellular medium. The cell pool of inorganic P and the uptake of PO(4) from the medium are low in K-limited cells and are immediately stimulated by the addition of K, suggesting that the primary effect of K limitation is to inhibit PO(4) uptake. All the metabolic effects of K limitation can be attributed to inhibition of PO(4) uptake. The requirement of extracellular K for PO(4) uptake may be due to a coupling between the uptake of K and PO(4).