Studies of the congenitally goitrous sheep. Iodoproteins of the goitre

1. Congenitally goitrous thyroid tissue was obtained from South Australian Merino sheep. Ultrastructural studies of the secretory cells in this tissue showed active cells of normal appearance, containing apical protein droplets. 2. (125)I-labelling in vivo of goitre tissue was used to investigate the iodoproteins, in which the major proportion of (125)I appeared in the cell protein fraction soluble in 0.9% sodium chloride (average 62% in goitres from untreated sheep). 3. Ammonium sulphate fractionation showed two clear peaks of iodoprotein precipitation, one at 35-40% saturation and the other at 50-55% saturation. Both iodoprotein fractions contained iodotyrosines and iodothyronines, which were identified chromatographically after enzymic hydrolysis of the protein. 4. Polyacrylamide-gel electrophoresis at pH9.4, at either 7.5 or 5.0% acrylamide concentration, was used to characterize the iodoproteins. Two major fractions were observed, the fastest-migrating fraction coincident with serum albumin, and a slower-migrating, less-well-defined zone. This fraction migrated in 7.5% acrylamide gel, which excluded normal thyroglobulin. 5. Density-gradient (10-40% sucrose) centrifugation was used to determine the approximate sedimentation coefficients of the iodoproteins, which showed major components at s(20,w) 8-9S and s(20,w)<5S. 6. Immunoprecipitation with rabbit anti-(sheep thyroglobulin) failed to sediment (125)I-labelled proteins from goitre extracts. 7. Ouchterlony-type double diffusion in agar plates demonstrated immunoprecipitation lines between rabbit anti-(sheep thyroglobulin) and both the concentrated goitre extract and its Sephadex G-200-excluded fraction, which were confluent with that obtained on reaction with purified normal thyroglobulin. 8. It was concluded that both major iodoprotein fractions were capable of supplying thyroid hormones to the animal, and that the fraction of s(20,w)<5S was iodinated serum albumin. As (125)I-labelled thyroglobulin was not detected in goitre tissue from untreated or thyroxine-treated animals, it was possible that the genetic defect causing goitre resulted in an abnormal thyroglobulin, incapable of being iodinated but immunologically reactive.     

Is there a double pathway for the secretion of thyroglobulin: a "short circuit" weakly glycosyl-iodinated and a "long circuit" strongly glycosyl-iodinated?

Intact rat thyroid lobes incubated in vitro release recently synthesized thyroglobulin (Tg) into the media at a faster rate than they release thyroglobulin stored in follicular structures. Differential release of this Tg fraction cannot be explained by morphological alterations in thyroid architecture during incubation. This rapidly excreted fraction exhibits a low density on rubidium chloride gradients characteristic of poorly sialylated and poorly iodinated thyroglobulin, comigrating on rubidium chloride gradients with thyroglobulin isolated from tunicamycin treated glands. This poorly sialylated and poorly iodinated thyroglobulin is itself unaffected in its density or release into the media by tunicamycin treatment. Tg isolated from the media of tunicamycin treated glands has nearly the same low iodine and low sialic acid content as rat serum thyroglobulin and does not incorporate radiolabelled glucosamine. This fraction thus appear to duplicate properties of low glycosylated-low iodinated thyroglobulin released from thyroid cells in organisms that have no follicular structures and no follicular storage process as well as from thyroid tissue in patients with thyroid disease states, particularly thyroid tumors. Thus it is proposed a "short loop" pathway of low-glycosylated low-iodinated thyroglobulin directly into circulation, that bypasses and is not stored in the follicular lumen, the "long loop".     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine: a microgel electrophoretic study of single rat thyroid follicles

Prophylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis. It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks. A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine A microgel electrophoretic study of single rat thyroid follicles

Propylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis.It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks.A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

Hydrolysis of 125I-labelled thyroglobulin by pancreatin, Pronase and pepsin

1. Pancreatin or Pronase hydrolysis of partially purified heat-denatured ¹²⁵I-labelled thyroglobulin does not release all the iodoamino acids from peptide linkage. 2. The addition of thyroidal proteases is essential to obtain maximum hydrolysis of thyroglobulin. 3. Pronase plus a fresh non-radioactive thyroid homogenate gives the best hydrolysis results, but peptide `mapping' shows that about 10% of the radioactivity remains in the form of peptides. 4. These peptides have approximately the same R_F as the iodotyrosines on one-dimensional chromatograms developed in butan-1-ol–2n-acetic acid (1:1, v/v). 5. Peptic hydrolysis of thyroglobulin releases virtually all of the iodothyronines but little of the iodotyrosines, in contrast with the action of pancreatin. 6. Hydrolysis of thyroglobulin with pepsin followed by Pronase plus a source of thyroidal proteases is satisfactory, but the results are not superior to those obtained by using Pronase plus a source of thyroidal proteases.     

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines

Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. In conclusion: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ ions in a reconstituted Ca2+ATPase pump.

1. During purification of the Ca2+ATPase from sarcoplasmic reticulum of rabbit muscle, different fractions with similar Ca2+ATPase activity were found to vary greatly in their ability to catalyze 45Ca2+ translocation in reconstituted liposomal systems. 2. A heat-stable fraction isolated from the fraction most active in Ca2+ translocation enhanced several-fold the Ca2+ translocation rate of the least active fraction. It also increased the ratio of Ca2+ translocation to ATP hydrolysis over 5-fold. The properties of the coupling factor resemble those of the proteolipid previously described by MacLennan et al. (MACLENNAN, D.H., YIP, C. C., ILES, G. H., and SEAMAN, P. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 469-478). 3. When the heat-stable factor was added to either sarcoplasmic reticulum fragments or to liposomes after, rather than before, reconstitution, it acted as an ionophore abolishing Ca2+ translocation.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Isolation of pig thyroid lysosomes. Biochemical and morphological characterization.

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, beta-galactosidase and beta-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.     

Number and types of peptide chains in thyroglobulin: tryptic peptides of noniodinated hog thyroglobulin

In order to identify the number and types of peptide chains in thyroglobulin, noniodinated 19-S thyroglobulin obtained from goitrogen-treated hogs was exhaustively digested with trypsin (EC 3.4.4.4) after reduction and S-carboxymethylation. The digestion mixture was preliminarily separated into 30 fractions on Sephadex G-100 or G-15 and SE-Sephadex columns. The number of various tryptic peptides contained in each fraction was determined on peptide maps, where spots were detected with ninhydrin for total peptides and with each specific reagent for arginine, histidine or tyrosine-containing peptides. The number of total peptides observed in most of the fractions was estimated to be half the number of lysine plus arginine residues found in each fraction per mole of thyroglobulin, and the number of specific peptides was also close to half the number of each specific amino acid. These findings imply that thyroglobulin has 2-fold symmetry in the structure at the level of tryptic fragments and thus probably at the level of intact peptide chains.     

The identification of thyroglobulin within a rat thryroid subcellular fraction

A particulatte fraction obtained from a rat thyroid homogenate by centrifugation at 27 000 × g was compared to the soluble fraction used for the preparation of thyroglobulin. By sodium dodecyl sulfate polycrylamide gel electrophoresis analysis after reduction, the soluble fraction inculded, mainly, three high molecular weight componenents which became strongly radioactive after an in vivo radioiodine injection. The particulate fraction on the same gels after reduction inlcude several low molecular weight components and a single high molecular weight component containing little radioiodine and showing, by electron microscopy, at least three types of structure: rough surfaced and smooth surfaced vesicles and apical vesicles (the secretory granules of thyroid follicular cells). The substrate present in the particulate fraction behaved antigenically like thyrolobulin. It is concluded that the particulate fraction contained nascent thyroglobulin inits undegraded form and that this material is probably the precursor to the various components encountered in the soluble fraction.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.     

Hydrolysis of insoluble cellulose to glucose catalyzed by cellulase‐containing liposomes in an aqueous solution of 1‐butyl‐3‐methylimidazolium chloride

The liposome containing cellulase from Trichoderma viride was prepared under the condition that an appreciable amount of cellulase was incorporated in lipid membranes. The liposomal cellulase and free enzyme were examined in their hydrolytic activities to insoluble cellulose powder CC31 in the acetate buffer solution (pH 4.8) of 15 w/w% [Bmim][Cl] (1‐butyl‐3‐methylimidazolium chloride). The mean diameter and size distribution of cellulase‐containing liposome were practically unchanged under the above condition. The free cellulase was deactivated more rapidly than the liposomal cellulase in catalyzing the hydrolysis of 2.0 g/l CC31 at 45°C in the presence of [Bmim][Cl] for 48 h. The activities of liposomal and free cellulase to cellobiose as soluble substrate were less susceptible to [Bmim][Cl] than their cellulolytic activities to CC31, meaning that β‐glucosidase is relatively stable among the three enzyme components of cellulase. The rate of glucose production could be appreciably improved by the pretreatment of CC31 with [Bmim][Cl] alone at 120°C for 30 min followed by the liposomal cellulase‐catalyzed hydrolysis of the substrate at 45°C at the [Bmim][Cl] concentration of 15 w/w%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1190–1196, 2013     

Acyl-coenzyme A: cholesterol acyltransferase. Transfer of cholesterol to its substrate pool and modulation of activity

The preincubation at 37 degrees C of rat liver microsomal fraction, followed by re-isolation of the treated vesicles, results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The presence of cholesterol-phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration-dependent. The preincubation of the microsomal fraction in the presence of [3H]cholesterol-phospholipid liposomes results in transfer of [3H]cholesterol to the re-isolated microsomal vesicles and this transfer follows first-order kinetics in respect to the donor concentration. These preincubations result also in a time-dependent and liposomal cholesterol concentration-dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half-time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltransferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time-dependent and temperature-dependent increase in cholesterol acyltransferase activity is due to the transfer of non-esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.     

Secretion of sulfated thyroglobulin

Thyroid follicle cells from various mammalian species incorporate 35-SO4(2-). Light and electron microscopic autoradiographs show that the Golgi complex is the predominant site of sulfate incorporation and that the secretory product accumulating in the follicle lumen is sulfated. In order to determine which components of the luminal content carry the sulfate residues, inside-out follicles from pig thyroid glands were incubated in the presence of 35-SO4(2-) and the secretory product released into the culture medium was analyzed by polyacrylamide gel electrophoresis. The observations show that the secretory product consists of sulfated thyroglobulin and that approximately 13 sulfate residues are bound covalently to 1 molecule of dimeric thyroglobulin. Digestion of 35-SO4(2-)-thyroglobulin with endoglycosidase H removes 20 to 30% of the radioactivity, indicating that the high mannose carbohydrate side chains carry sulfate residues. The complex carbohydrate side chains are apparently free of sulfate since treatment with endoglycosidase D did not alter the sulfate content. About 2/3 of the sulfate is cleaved by hydrolysis with 1 M HCl (5 min, 95 degrees C) indicating the presence of tyrosine sulfate. Part of the sulfate is exposed and presumably located on the surface of the thyroglobulin molecule as suggested by the direct accessibility of 35-SO4(2-)-thyroglobulin to digestion with sulfatases. The sulfate residues contribute to the anionic state of thyroglobulin. It is postulated that the sulfate residues operate in the regulation of thyroglobulin transport in the cell and in the tight packaging of thyroglobulin in the follicle lumen.     

The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA: Cholesterol acyltransferase substrate pool in liver microsomal fraction

The incubation at 37°C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA: cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these values was also the activation energy for the increase in acyl-CoA: cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.     

Evidence for the hydrolysis of topical applied liposomal lipids in human stratum corneum

Liposomes were incubated with an extract of human plantar stratum corneum. The liposomal lipids were hydrolysed, if composed of soy-bean phosphatidylcholine or phosphatidylglycerol. Rigid lipids were not degraded. The temperature optimum of the hydrolysis was between 30-35degC. CaCl enhanced, while EDTA reduced the rate of hydrolysis, indicating that the hydrolysis is due to a phospholipase A(2).