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Biomedical subjects

M G Wetzel

Publications and source records attributed to M G Wetzel.

At least 19 recordsLinked to original sources
PubMed1998

Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay.

The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.

Adult↗
PubMed1995

Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembly.

Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.

Animals↗
PubMed1995

Soluble-binding proteins for docosahexaenoic acid are present in neural retina.

PURPOSE: To determine if soluble binding proteins (BP) for fatty acids (FA), particularly docosahexaenoic acid (DHA), could be identified in the cytosol of rat and bovine retinas under in vitro and in vivo conditions. METHODS: In vitro, cytosol fractions from normal bovine and rat retinas were delipidated and incubated with [14C]-DHA with or without a number of competing fatty acids. After crosslinking bound FA to BP, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity determined in each fraction. For in vivo experiments, rats received [14C]-DHA by gavage. At selected periods after ingestion, retinas were collected and cytosolic fractions were prepared from each. These were crosslinked and subjected to SDS-PAGE, and radioactivity was determined in each fraction. RESULTS: In vitro, several peaks of radioactivity (approximately 13, 20, 32, 43-45, 50, 63, and 94-105 kd) were found that exhibited [14C]-DHA binding. Relative specificity of binding was assessed by blocking of the [14C]-DHA binding with unlabeled DHA and by competition experiments with other FAs. After in vivo ingestion of [14C]-DHA, a large peak of radioactivity was observed at 43 kd by 4 hours. At 6 hours, this peak decreased and, after 24 hours, it approached baseline. CONCLUSIONS: These findings demonstrate that there is a discrete grouping of proteins in retinal cytosol capable of binding DHA and other FA. Although the identities of these proteins have yet to be determined, the group may include a member of the 12- to 15-kd group of small fatty acid binding proteins (FABP). In particular, a unique 43-kd binding peak could play a major role in the uptake, binding, or both of DHA by the retina in vivo.

Animals↗
PubMed1994

Transport of phosphatidylcholine to Xenopus photoreceptor rod outer segments in the presence of tunicamycin.

Study of the dynamics of membrane protein and phospholipid transport from the inner to the outer segment of vertebrate photoreceptors has shown an interesting dissociation of the two components under a number of experimental treatments which inhibit protein synthesis or transport. Under conditions which block the addition of opsin to outer segments, various lipids continue to be synthesized and transported to the outer segment in the presence of monensin, puromycin, brefeldin A, tunicamycin and several general metabolic inhibitors. In the current study, isolated retinas from adult Xenopus laevis were incubated with or without 20 micrograms mg-1 of tunicamycin in total darkness or light for 2-12 h in the presence of [3H]choline to study the dependence of phosphatidylcholine synthesis and transport on protein transport to the outer segment. Phosphatidylcholine is a major bulk lipid of outer segments, comprising close to one half of the phospholipid of outer segment phospholipids, and blocking choline uptake in retinas is known to cause photoreceptor degeneration. Biochemical analysis demonstrates that tunicamycin does not block the synthesis of phosphatidylcholine in photoreceptor inner segments or transport of radiolabelled phosphatidylcholine to outer segments during 6 h incubations with [3H]choline in light or total darkness. Light and electron microscopic autoradiography and morphometric analysis show that [3H]choline radiolabelled phospholipid does not accumulate in a band of newly formed basal discs in the outer segment or in the tubulo-vesicular structures which accumulate in the intersegmental space of tunicamycin-treated retinas. We conclude that transport of phosphatidylcholine can occur independently of opsin transport to the outer segment but whether this represents two separable components of a single pathway or involves two distinct routes of transport to the outer segment is still unresolved.

Animals↗
PubMed1993

Tunicamycin does not inhibit transport of phosphatidylinositol to Xenopus rod outer segments.

Tunicamycin inhibits the dolichol pathway for N-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. Adult Xenopus retinas were preincubated in dim red light with 20 micrograms ml-1 of tunicamycin for one hour followed by incubation in the light for 2-6 h with tunicamycin plus either [3H]mannose, [3H]leucine, [2-(3)H]glycerol or [3H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [3H]leucine and [3H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [3H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [3H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [3H]glycerol and [3H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.

Animals↗
PubMed1992

Transport of n-3 fatty acids from the intestine to the retina in rats.

This study was undertaken to determine the mode of transport of the essential (n-3) fatty acids docosahexaenoic acid 22:6(n-3) and linolenic acid 18:3(n-3). Male weanling Sprague-Dawley rats received a mixture of corn oil and [14C]18:3(n-3) or [14C]22:6(n-3) by gavage. At periods of 1 to 4 days after the injection, four rats per time point were killed and samples of blood were taken via heart puncture and the livers and retinas were collected. Blood lipoproteins and plasma proteins were separated by ultracentrifugation and analyzed by HPLC. Lipids were extracted and saponified and the fatty acids were converted to phenacyl esters for separation of individual fatty acids. After 1 and 2 h, radioactivity from 18:3(n-3) and 22:6(n-3) was observed primarily in the chylomicron/very low density lipoprotein fraction. By 4 h, radioactivity in the lipoprotein fraction was greatly decreased, with a small amount of radioactivity associated with albumin in the soluble protein fraction. After 24 h, the total amount of radioactivity associated with lipoprotein was further reduced, with more than half of the remaining label occurring in association with albumin and another unidentified protein. In the liver, 22:6(n-3) was concentrated in triacylglycerols (40.7%) and phospholipids (51.1%), with a maximum specific activity at 4 h. In the rod outer segments (ROS), the specific activity of [14C]22:6(n-3) increased to a maximum at 24 h and maintained a high level even at 4 days. These data suggest that after injection, 18:3(n-3) and 22:6(n-3) are esterified to triglyceride and phospholipid by the intestinal absorptive cells and transported in chylomicrons to the liver. After conversion of 18:3(n-3) to 22:6(n-3) in the liver, the retina accumulates 22:6(n-3) which may be transported from the liver via albumin and another unidentified protein, and is retained by the rod outer segments.

Animals↗
PubMed1991

Metabolism of linolenic acid and docosahexaenoic acid in rat retinas and rod outer segments.

Docosahexaenoic acid (22:6 omega 3) is uniquely enriched in photoreceptor outer segment phospholipids, comprising up to one-half of the fatty acids of phosphatidylethanolamine and phosphatidylserine. The current study was designed to investigate the incorporation of 22:6 omega 3 into outer segment phospholipids over 12 days and to determine whether the retina contained the enzymes necessary for elongation and desaturation of the major dietary precursor of 22:6 omega 3, the essential fatty acid linolenic acid (18:3 omega 3). Sprague-Dawley rats were injected intravitreally with [14C]22:6 omega 3 or [14C]18:3 omega 3 and kept in cyclic light (12 hr light/12 hr dark) for 2 hr to 12 days. Phospholipids from rod outer segments and the remaining retinal debris were separated by two-dimensional thin-layer chromatography. [14C]22:6 omega 3 radioactivity was initially highest in phosphatidylcholine and rapidly decreased from 45% of total phospholipid labeling at 2 hr to 26% by 1 and 3 days in ROS, while phosphatidylethanolamine labeling increased from 49 to 68% by 3 days and phosphatidylserine labeling increased from 3 to 14% over 12 days. Phenacyl derivatives of total fatty acids were separated by HPLC. A substantial conversion of [14C]18:3 to [14C]20:5, [14C]22:5 and [14C]22:6 was noted after 1 days, with increasing conversion to [14C]22:6 over the 12-day period. When only one eye was injected with [14C]18:3 omega 3, negligible radioactive fatty acids were detected in the contralateral eye from 1 to 12 days post-injection demonstrating that conversion of 18:3 to 22:6 occurred primarily within the injected eye. All enzymes for elongation and desaturation of 18:3 to 22:6 appear to be present in the eye. However, the conversion of 22:5 to 22:6 by delta-4 desaturase is evidently rate-limiting and may affect phospholipid replacement during photoreceptor outer segment renewal if this pathway proves to be essential for the supply of 22:6 during disk membrane formation.

Animals↗
PubMed1990

Metabolic labeling of rod outer segment phospholipids in miniature poodles with progressive rod-cone degeneration (prcd).

The recessive genetic defect in miniature poodles which results in progressive rod-cone degeneration (prcd) has been investigated in an attempt to determine the biochemical abnormality involved. In the present study, the rod outer segments of young prcd affected miniature poodles and normal dogs have been compared with respect to the incorporation of intravitreally injected [3H]palmitic acid. [14C]linolenic acid, and [14C]docosahexaenoic acid into neutral lipids and phospholipids as well as [3H]palmitate and [14C]leucine into rhodopsin. In addition, 3 mm trephined punches of retinas were incubated with [3H]palmitic acid, [3H]arachidonic acid, [14C]linolenic acid, [3H]serine, [14C]glycerol and [14C]leucine. No difference in incorporation of labeled precursors into lipids or rhodospin was noted between prcd affected and normal retinas. Phosphatidyl choline appeared to function as a carrier of fatty acids to the rod outer segment where they were redistributed to other phospholipids. An interesting lack of conversion of the essential fatty acid linolenic acid to docosahexaenoic acid was noted in both normal and affected retinas. This conversion involves elongation and desaturation of linolenic acid and may take place primarily in extraretinal tissues such as the liver. This finding, in conjunction with a parallel study of plasma fatty acids which has shown significantly lower levels of docosahexaenoic acid in prcd affected poodles, points to a possible systemic defect in the metabolism or transport of docosahexaenoic acid, a fatty acid uniquely enriched in the photoreceptor outer segments.

Animals↗
PubMed1989

Metabolic labeling of normal canine rod outer segment phospholipids in vivo and in vitro.

Twenty-four hours after the intravitreal injection of [3H]palmitate and [14C]docosahexaenoate in dogs, the rod outer segment phospholipids are highly labeled. Palmitate is found predominantly in phosphatidylcholine (PC), with lesser amounts in phosphatidylethanolamine (PE) and very little in either phosphatidylserine (PS) or phosphatidylinositol (PI). Docosahexaenoate most heavily labeled PE followed by PC, with lesser amounts in PS and very little in PI. Two-hour incubations of 3 mm trephine buttons removed from dog retinas produced very similar patterns of labeling with palmitate and docosahexaenoate. In vitro incubation of retina buttons with [3H]arachidonate produced heavy labeling of PI, with much less in PC and very little in either PS or PE. [3H]Glycerol labeled in PC, PI and PE in descending order but PS almost not at all. [3H]Serine labeled PS predominantly, but small amounts were found in PC, PE and PI. The trephine retina buttons can be utilized for multiple-precursor incubations and studies of differential metabolism in retinal regions, particularly when studying scarce tissue from mutant animals or humans with inherited retinal degenerations.

Animals↗
PubMed1989

Fatty acid metabolism in normal miniature poodles and those affected with progressive rod-cone degeneration (prcd).

It is possible that a genetic defect observed in the prcd poodle involves the abnormality of an enzyme which functions in phospholipid or lipoprotein metabolism. In our studies thus far, we have been unable to detect any defect in retinal phospholipid biosynthesis, but we have noted a decrease in plasma levels of 22:6w3 which may be a result of an enzyme defect in liver biosynthesis of 22:6w3 from its dietary precursor, linolenic acid, or some defect in the blood lipoprotein transport of this essential fatty acid. If 22:6w3 is essential to the normal elaboration and functioning of the photoreceptor outer segment, it is possible that decreased access to this fatty acid due to lower blood levels of 22:6w3 could cause photoreceptor abnormalities. Further studies are needed to confirm the possible defect in delta-4 desaturase activity and possible dietary modification of the course of this prcd retinal degeneration.

Animals↗
PubMed1986

Turnover of palmitate, arachidonate and glycerol in phospholipids of rat rod outer segments.

Rat retinas were intravitreally labeled with [3H]palmitic acid, [3H]arachidonic acid or [3H]glycerol to study the turnover of the component parts of the major phospholipids in rod outer segments at times ranging from 2 hr to 12 days post injection. Rod outer-segment and retinal debris fractions were extracted and the major phospholipids separated by two-dimensional thin-layer chromatography. In darkness, [3H]glycerol rapidly labeled phosphatidylinositol in both rod outer-segment and retinal debris fractions. The label in phosphatidylinositol subsequently decreased dramatically, demonstrating a rapid turnover of phosphatidylinositol with a half-life of less than 1 day. Phosphatidylcholine and phosphatidylethanolamine were maximally labeled by glycerol in the retinal debris at the 2-hr time-point and were maximally labeled in rod outer segments between 1 and 5 days post injection, with somewhat longer residence times in the rod outer segments. Phosphatidylserine showed a lag in initial labeling in both rod outer-segment and retinal debris fractions indicating that this phospholipid is not a major precursor of phosphatidylcholine and phosphatidylethanolamine in rat retinas. [3H]Palmitate and [3H]arachidonate labels were rapidly incorporated into outer-segment phospholipids by 1-2 hr post injection. Eighty per cent of the palmitate label was initially associated with phosphatidylcholine at 2 hr. The total amount of palmitate label in rod outer-segment phosphatidylcholine did not change for 12 days post injection. Outer-segment phosphatidylethanolamine steadily increased in palmitate label throughout the 12-day period, suggesting that phosphatidylethanolamine may be utilized for recapture of palmitate released from breakdown of palmitate esters of rhodopsin or vitamin A or from phospholipids. Arachidonate primarily labeled phosphatidylinositol and phosphatidylcholine of both rod outer segments and retinal debris. The arachidonate label did not decrease dramatically during the first day in phosphatidylinositol as did the glycerol label, indicating that arachidonic acid is reutilized by the retina. Turnover of the individual phospholipids, as measured by a decrease in glycerol labeling of the phospholipid backbone, is more rapid than the loss of palmitate label, indicating that there is extensive reutilization of palmitate in both phosphatidylcholine and phosphatidylethanolamine of the rod outer segment. The data indicate that palmitate derived from many sources could be used by the photoreceptor to acylate rhodopsin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Effect of the combined lipase deficiency mutation (cld/cld) on ultrastructure of tissues in mice. Diaphragm, heart, brown adipose tissue, lung, and liver.

Lipoprotein lipase and hepatic lipase activities are very low in tissues of mice born with genetic combined lipase deficiency (cld/cld). Consequently, if allowed to suckle, the mice develop severe hyperlipemia and die within 3 days. The ultrastructure of capillaries and parenchymal cells in tissues that normally contain lipoprotein lipase and hepatic lipase was studied in tissues from cld/cld and unaffected mice 6 to 24 hours of age. Capillaries in tissues from suckled cld/cld mice were packed with numerous abnormally shaped chylomicrons. There was close contact between surfaces of chylomicrons and the luminal plasma membrane of endothelium. Chylomicrons were sometimes found between endothelial cells and in the subendothelial space in heart, lung, and liver, and in the lumen of lung alveoli. In contrast, capillaries of suckled unaffected mice contained very few chylomicrons, and the subendothelial spaces and lung alveoli were free of chylomicrons. Myocytes of diaphragm and heart from suckled cld/cld mice did not contain lipid droplets, whereas brown adipocytes contained a few small droplets. Parenchymal cells in diaphragm, heart, brown adipose tissue, and lung from suckled unaffected mice contained numerous large lipid droplets. Hepatocytes of suckled cld/cld mice contained small irregularly shaped lipoprotein particles (100 A) in endoplasmic reticulum and Golgi, numerous large lysosomes containing small lipoprotein particles, lipid spheres and lamellar structures, and no intracellular lipid droplets, whereas hepatocytes of suckled unaffected mice contained larger lipoprotein particles (400 A), large lipid droplets, and very few lysosomes. Triacylglycerol of chylomicrons from cld/cld mice was readily hydrolyzed by bovine lipoprotein lipase in vitro, and this effect was not augmented by heat-inactivated serum, indicating that the chylomicrons contained adequate amounts of apoprotein C-II. Thus, the large amount of chylomicrons in capillaries and small amount of lipid droplets in cells of suckled cld/cld mice reflect the very low level of lipoprotein lipase activity in these animals. The findings in hepatocytes indicate that lipoprotein metabolism in liver is markedly disturbed in cld/cld mice.

Adipose Tissue, Brown↗
PubMed1984

Lipolysis and fatty acid transport in rat heart: electron microscopic study.

Lamellar structures with a periodicity of 50 A developed in myocytes of glutaraldehyde-fixed heart tissues from young and adult rats when the tissues were incubated with tannic acid (pH 7.4) at 25 degrees C. The increase in lamellar structures (P less than 0.025) was accompanied by a significant decrease in intracellular lipid droplets (P less than 0.025), indicating that tissue lipase was active in fixed tissue and that the lamellar structures were probably composed of fatty acids formed by lipolysis. The lamellar structures in myocytes were located in the lumen of intracellular channels near lipid droplets and mitochondria and in the outer compartment of mitochondria. Lamellar structures were found at the periphery of chylomicrons, in intraendothelial channels, and in extracellular space of incubated fixed tissues from chylomicron-injected young rats. Chylomicron-lipid disappeared from capillaries (P less than 0.025) and lamellar structures with wide interlamellar spacings (80-1,000 A) developed in the extracellular space surrounding capillaries (P less than 0.025) in unfixed heart tissue from chylomicron-injected fasted young rats when the tissue was incubated without tannic acid; lamellar structures did not develop in similarly treated tissue from uninjected rats. Thus the lamellar structures found in extracellular space represent fatty acids derived from lipolyzed chylomicrons. We conclude that fatty acids produced by lipolysis in incubated heart accumulated and spread in an interfacial continuum of external leaflets of cell membranes extending from the capillary lumen to extracellular space and from intracellular lipid droplets to the interior of mitochondria in myocytes. When fatty acids overcrowded the continuum, they formed lamellar extensions of the continuum at different sites along its course through the tissue.

Aging↗
PubMed1982

Transfer of human lymph chylomicron constituents to other lipoprotein density fractions during in vitro lipolysis.

To ascertain whether chylomicron constituents would be transferred to low density lipoprotein (LDL, d 1.019-1.063 g/ml) and high density lipoprotein (HDL, d 1.063-1.21 g/ml) density fractions during lipolysis in the absence of other lipoproteins, the in vitro effect of bovine milk lipoprotein lipase on human thoracic duct lymph chylomicrons in the presence of albumin was examined. In incubations without lipase, over 90% of chylomicron constituents remained in the 1.006 g/ml supernate, and large particles ranging in diameter mainly from 750-6000 A were observed by electron microscopy. After the addition of lipase, lipolysis ranged from 69.0-94.6% and numerous collapsed particles with redundant surface were seen, as well as smaller particles within the LDL and HDL density region. With lipolysis, the majority of chylomicron cholesterol and phospholipid mass was transferred to LDL and HDL, while chylomicron apolipoprotein (apo) A-I, A-II, and C-II mass was transferred mainly to HDL. Utilizing either radioiodinated apoA-I and apoA-II reassociated with chylomicrons or radiolabeled chylomicrons, a similar redistribution of apoA-I and apoA-II radioactivity was noted with lipolysis. In contrast, chylomicron apoB (mainly B-48) radioactivity was transferred predominantly to LDL with lipolysis. These data are consistent with the concept that during lymph chylomicron triglyceride hydrolysis, chylomicron apolipoproteins, cholesterol, and phospholipid can be transferred to the LDL and HDL density regions in the absence of acceptor particles.

Animals↗
PubMed1971

Plasma and phagosome membranes of Acanthamoeba castellanii.

Plasma membranes were isolated from the ameba Acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. The isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). The plasma membranes had very low concentrations of DNA, RNA, lipid inositol, and glycerides. Glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the whole cell. The plasma membranes were also judged to be of high purity by the absence, or very low level, of enzymatic activities considered to be indicative of other cell membranes, and by electron microscope examination. Alkaline phosphatase and 5'-nucleotidase activities were enriched in the plasma membranes 13-fold relative to the whole homogenate and had higher specific activities in the plasma membranes than in any other cell fractions. A Mg(++) adenosine triphosphatase (ATPase) was enriched sixfold in the plasma membranes relative to the whole homogenate. The phospholipids of the plasma membranes contained more phosphatidylethanolamine and phosphatidylserine and less phosphatidylcholine than did the phospholipids of the whole cells. There were differences in the fatty acid compositions of corresponding phospholipids in the plasma membranes and whole cells but no difference in the ratios of total saturated to unsaturated fatty acids. The membranes of phagosomes isolated from amebae that had ingested polystyrene latex had essentially the same phospholipid, sterol, and enzymatic composition as plasma membranes.

Adenosine Triphosphatases↗
PubMed1969

Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes.

A method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of Acanthamoeba castellanii (Neff) that have been incubated with polystyrene latex beads. Cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. Identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays of acid phosphatase, alpha- and beta-glucosidase, and reduced nicotinamide adenine dinucleotide dehydrogenase. When phagocytosis was allowed to occur for longer periods the phagocytic vesicles appeared to fuse with each other and perhaps with digestive vacuoles. The resultant vesicles which contained many beads were heavier than those which consisted of only one bead or a few beads with a closely applied membrane. Ultrasonication ruptured the isolated vesicles, and the membranes could then be isolated in 30-50% yield based on phospholipid analysis. These membranes were essentially free of acid hydrolases and, presumably, other soluble proteins, as was also indicated by their low ratio of protein to phospholipid. The membranes have been prepared both as closed vesicles and as open sheets.

Acid Phosphatase↗