Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves.

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.

UDP-glucose:solasodine glucosyltransferase from eggplant (Solanum melongena L.) leaves: partial purification and characterization.

Uridine 5'-diphosphoglucose-dependent glucosyltransferase which catalyzes the glycosylation of solasodine i.e. UDP-glucose:solasodine glucosyltransferase, is present in leaves, roots, unripe fruits and unripe seeds of eggplant (Solanum melongena L.). The glucosylation product is chromatographically identical with authentic solasodine 3 beta-D-monoglucoside, a putative intermediate in the biosynthesis of solasodine-based glycoalkaloids characteristic of the eggplant. The enzyme was purified about 50-fold from crude cytosol fraction of eggplant leaves by ammonium sulphate precipitation and column chromatography on Q-Sepharose and Sephadex G-100. The native enzyme has a molecular mass of approx. 55 kDa and pH optimum of 8.5. Divalent metal ions are not required for its activity but the presence of free-SH groups is essential. Besides solasodine (Km = 0.04 microM), the enzyme effectively glucosylates tomatidine, another steroidal alkaloid of the spirosolane type, but it is virtually inactive towards the solanidane-type steroidal alkaloids such as solanidine or demissidine. The enzyme is specific for UDP-glucose (Km = 2.1 microM) since unlabelled ADP-, GDP-, CDP- or TDP-glucose could not effectively compete with UDP-[14C]glucose used as the sugar donor for solasodine glucosylation. Moreover, no synthesis of labelled solasodine galactoside was observed when UDP-[14C]glucose was replaced with UDP-[14C]galactose.

Characterization of acyllipid: sterol glucoside acyltransferase from oat (Avena sativa L.) seedlings.

Membranous fractions from leaves of oat seedlings readily convert cholesterol beta-D-glucoside into its 6'-O-acyl derivative using endogenous acyllipids as acyl sources. Experiments with delipidated enzyme preparations showed that among acyllipids present in oat leaves digalactosyldiacylglycerols are evidently the best acyl donors in this reaction. Beside of sterol glucosides, the enzyme can acylate beta-D-glucosides of several other steroids, although at very different rates.

An experimental skin sandwich flap on an independent vascular supply for the study of percutaneous absorption.

Further insights into the composite interactive processes of topically applied agents and percutaneous absorption and metabolism by functional skin in vivo have been hampered by the lack of a model system wherein the blood flow to and from the skin is independent but experimentally accessible. Utilizing microsurgical techniques, split-thickness skin grafting with syngeneic skin grafts, and the congenitally athymic (nude) rat, a skin sandwich flap system has been generated that has an independent but accessible vasculature and thus fills this void. We describe the methodology that has been developed to create the flap and present experiments that: demonstrate a lack of significant collateral circulation; quantify the microcirculation of the skin sandwich flap, host side, and graft side at various times during and after the flap has been generated, and note that blood flow to the flap is basically unchanged from host skin; demonstrate the utility of the system in measuring the amount of [14C]benzoic acid that appears in the flap when deposited on the surface in volatile and nonvolatile vehicles as a function of time; and demonstrate the fact that the flap can be reused, and that the total amount of [14C]benzoic acid absorbed across skin does not change in a substantial way as the flap ages.

Description of and treatment to inhibit the rejection of human split-thickness skin grafts by congenitally athymic (nude) rats.

Gradual rejection of topically engrafted human split-thickness skin grafts (HSTSG) occurred in greater than 90% of congenitally athymic (nude) rats between 21 and 42 days of grafting. Engraftment and rejection of HSTSG is accompanied by a partial restoration of some cell-mediated immune components, the mixed lymphocyte response and lysis of human target cells. Histologic features of the rejection process were those seen in a host-versus-graft reaction. Immunofluorescent analysis of skin undergoing rejection demonstrated IgG at the basement membrane zone in most grafts. Nude rats rejecting HSTSG had circulating IgG which bound to the basement membrane zone and blood vessels of human skin. Nude rats treated with cyclosporine injections for 21 days had an enhanced survival of HSTSG, 120 or more days.

The development of a rat/human skin flap served by a defined and accessible vasculature on a congenitally athymic (nude) rat.

Experience in microvascular surgery on rats and availability of athymic (nude) rats led us to believe that a long-term functional rat/human skin sandwich flap could be generated on a defined and experimentally accessible vasculature on nude rats. Such a system has been developed and validated. Microvasculature has been assessed. The volume of blood to the flap ranges from 1 to 2 ml/min, collateral circulation to the flap exists, but is negligible, and there is little change in the capillary blood flow as the flap ages. The flap can be utilized to study absorption of compounds from a half-cell diffusion chamber or from direct deposition on the skin, and can be utilized to study various parameters of percutaneous absorption, e.g., the effect of hydration on the stratum corneum. Transdermal flux can be determined. Altering the microcirculation directly affects the percutaneous absorption of compounds that are rapidly absorbed. The absorption of benzoic acid through an experimentally vasoconstricted area (iontophoresis of phenylephrine) significantly alters the time to peak absorption, with values being 14 times that of the control site. The system has been utilized to assess metabolic activity of skin in situ using [3H]adenine arabinoside and studying the appearance of its major metabolite, [3H]Ara-H, in flap blood, as well as the back diffusion of this compound into the donor chamber. Recently the human/rat skin sandwich flap component has been developed. With this system, it has been demonstrated that benzoic acid, when applied to the human skin component of the flap has an absorption profile which is quite different from that when benzoic acid is applied to rat skin, peak flux occurred 2 hr after application. This contrasts with 10 min to peak flux when the same experiment is carried out on the rat/rat skin sandwich flap. To our knowledge, the human/rat skin sandwich flap is the first example of a viable, functional human organ that is chronically maintained by a biologic support system which has the added distinction of being on an independent but accessible vasculature. The validation experiments strongly suggest that this system will be important in gaining insights into the more sophisticated in vivo components of skin, relative to toxicology and pharmacology.

Characterization of wax-ester hydrolase from roots of white mustard (Sinapis alba L.) seedlings.

The activity of wax-ester hydrolase in roots of white mustard (Sinapis alba L.) seedlings is located in a membranous fraction sedimenting at 15000 g. The enzyme which shows a high degree of hydrophobicity was solubilized with a synthetic detergent Triton X-100 and purified about 70-fold by acetone precipitation and gel permeation chromatography on Sepharose 6B. The purified enzyme preparation was active within a broad pH range of 5.8-8.5. Hydrolase activity with hexadecanyl palmitate as the substrate was stimulated by Triton X-100 and dithioerythritol. Of wax esters containing saturated fatty acids C2-C22 and saturated, primary alcohols C2-C24 the highest rate of hydrolysis was found with the esters containing palmitic acid (C16) and tetradecanol (C14). Data presented suggest that wax esters and steryl esters are either hydrolyzed by different specific enzymes or that two enzymes are present of different specificity towards the two substrates.

Enzymatic synthesis of wax esters by cell-free preparations from Sinapis alba L roots.

Acetone powder preparations from the roots of white mustard (Sinapis alba L) seedlings efficiently catalyse the esterification of [1-14C]n-hexadecanol in the presence of tripalmitoylglycerol as the fatty acid source. Free palmitic acid as well as mono- and dipalmitoylglycerols are much less effective which indicates that a direct acyl transfer from triacylglycerols to long-chain alcohols takes place. The formation of wax esters was also followed using tri-[1-14C]palmitoylglycerol and unlabelled alcohol. In this reaction straight-chain, saturated and unsaturated, primary alcohols as well as an isoprenoid alcohol--phytol can be utilized as acyl acceptors. Of the saturated straight-chain alcohols, C20 alcohol was esterified most efficiently. Triacylglycerols containing fatty acids C12-C22, both saturated and unsaturated, can serve as acyl sources. Among triacylglycerols containing saturated fatty acids trimyristoyl- and tripalmitoylglycerols are the best acyl donors.

Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum.

1. Sinapis alba L. seedlings contain glycosyltransferase catalyzing the synthesis of sterol glucosides in the presence of UDPglucose as sugar donor. The major activity occurs in the membranous fraction sedimenting at 300--9000 x g. Successive treatment of the particulate enzyme fraction with acetone and Triton X-100 affords a soluble glucosyltransferase preparation which can be partly purified by gel filtration on Sephadex G-150. Molecular weight of the glucosyltransferase is 1.4 . 10(5). Apparent Km values for UDPglucose and sitosterol are 8.0 . 10(-5) M and 5.0 . 10(-6) M, respectively. 2. Comparison was made of the S. alba glucosyltransferase with a similar sterol-glucosylating enzyme isolated from non-photosynthesizing organism Physarum polycephalum (Myxomycetes). UDPglucose was the most efficient glucose donor in both cases but the enzyme from Ph. polycephalum can also utilize CDPglucose and TDPglucose. Glucose acceptors are, in case of both enzymes, sterols containing a beta-OH group at C-3 and a planar ring system (5 alpha-H or double bond at C-5). The number and position of double bonds in the ring system and in the side chain, as well as the presence of additional alkyl groups in the side chain at C-24 are of secondary importance. 3. The present results indicate that both enzymes can be regarded as specific UDPglucose:sterol glucosyltransferases. Certain differences in their specificity towards donors and acceptors of the glucosyl moiety suggest, however, a different structure of the active sites in both enzymes.

Intracellular localization and some properties of UDPG: sterol glucosyltransferase from Calendula officinalis.

UDPG: sterol glucosyltransferase is localized in the 2-week-old C. officinalis seedling in the membrane structures, separated from chloroplasts and mitochondria, and consisting probably of fragments of the Golgi apparatus. A minor part of the enzyme activity is associated with the microsomal fraction. A number of synthetic detergents stimulate the activity of the membrane-bound enzyme causing its solubilization. The enzyme preparation purified about 70-fold is strongly inhibited by HgCl2 and p-chloromercuribenzoate; it is markedly stimulated by mercaptoethanol and dithiothreitol, and to a lesser extent by Mg2+ and Ca2+ as well as by some chelating and reducing agents. UMP stimulates and UDP and UTP markedly inhibit the enzyme activity. The enzyme does not act on 4-methylsterols although it utilizes a number of 4-demethylsterols. It seems that the presence of a double bond in ring B enhances the affinity of the substrate for the enzyme. Delta-5-Sterols are utilized at a higher rate than delta-7-sterols. Saturated sterols and delta-25-sterols are poor substrates.

S-adenosyl-L-methionine-cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus.

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine-cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C(28) and C(29) sterol production in the algae.