W. Howard Evans

The phospholipid and fatty acid composition of Schistosoma mansoni and of its purified tegumental membranes

The lipid compositions of mature male and female Schistosoma mansoni and cercariae were compared to that of the hepato-pancreas of unparasitised Biomphalaria globrata (the intermediate snail host), and of red blood cells and sera of hamsters (the mammalian host). Membranes were isolated from the tegument of mature schistosomes by spontaneous release into phosphate-buffered saline, with or without vortexing, and by removal from the parasites surface using poly-lysine beads. The phospholipid composition of the membranes prepared by the three methods showed a typical plasma membrane-like profile, with high sphingomyelin content (approximately 20%) and cholesterol to phospholipid molar ratio (0.7-1.1). The fatty acid compositions of the resolved phospholipid classes were analysed. Although the composition was in general unremarkable, a high content of eicosaenoic acid (20:1), rarely found in mammals, was noted in whole schistosomes, cercariae, the hepato-pancreas of unparasitised Biomphalaria, and the isolated tegumental membranes. Eicosaenoic acid was also found in adults of Schistosoma japonicum. Host serum lipids from normal and parasitised hamsters contained extremely low amounts of eicosaenoic acid, indicating that this fatty acid is probably continually synthesised by the parasite, probably from preformed fatty acid precursors provided by the host.

Purification and topographical location of tegumental alkaline phosphatase from adult Schistosoma mansoni.

Alkaline phosphatase, a marker for tegumental membranes of Schistosoma mansoni, was extracted using Triton X-100 from membranes purified by sucrose density gradient centrifugation. The enzyme activity was purified 6 800-fold over parasite homogenates and 118-fold over the tegumental membranes released when parasites were incubated in phosphate-buffered saline. Purification of the solubilised enzyme was achieved by binding to a Con A agarose affinity column, gel filtration of the eluted glycoproteins, and Blue affigel chromatography. The purified enzyme was shown to consist of a single glycosylated polypeptide Mr 65 000 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Enzyme activity was associated with a possible tetramer, Mr 260 000 on gel filtration. Activity was associated with a band Mr 130 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis run in the absence of reducing agents. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents, the size of the enzyme was shown to be similar in cercariae, schistosomula, adult schistosomes and their eggs, but it was smaller than the activity (Mr 145 000) extracted from host liver and intestinal microsomal membranes. The topography of the enzyme in the schistosome tegument was investigated by using surface radio-labelling reagents followed by its purification. The enzyme could be radio-iodinated only with difficulty in adult worms. Bolton and Hunter reagent, used at high concentration and for prolonged time periods, resulted in labelling of enzyme activity and the Mr 65 000 polypeptide subunit was also iodinated under these extreme conditions. It was concluded that the enzyme is not exposed at the schistomes surface, and is probably buried in the tegumental membrane network.

Biochemical evidence that Na+,K+-ATPase is located at the lateral region of the hepatocyte surface membrane

The secretion of bile by hepatocytes into the bile canaliculi is an active process accompanied by osmotic filtration of water and electrolytes [1,2]. The two major solutes are bile salts and sodium, and bile aciddependent and bile acid-independent components of bile formation are identified experimentally. Several lines of evidence suggest that bile acid-independent bile flow is regulated by a Na”,K’-ATPase located at the hepatocyte plasma membrane [3-61. The mammalian plasma membrane is, however, a complex organelle, and in hepatocytes three major domains are recognised anatomically [7] and physiologically [8,9]. It has been assumed in most studies that the Na’,K’-ATPase is located at the bile canalicular domain of the plasma membrane, and biochemical studies in which a single plasma membrane fraction containing mainly bile fronts was studied would appear to support this location [3-61. A more precise location for Na’,K’-ATPase activity can be identi~ed by using plasma membrane subfractions shown to originate from the sinusoidal, lateral and canalicular domains of the hepatocyte surface [ 12,231. This communication shows that Na+,K+-ATPase is located mainly in a plasma membrane subfraction characterised by intercellular junctions originating from the hepatocyte lateral side and that activity is low in a fraction containing high specific activities of a range of enzymes

Resolving biochemically the hepatocyte's multifunctional pericellular membrane

The hepatocytes pericellular membrane is complex and can be subfractionated to yield plasma membrane fractions from the blood sinusoidal, contiguous and bile canalicular regions. The differentiation of a plasma membrane into physiologically disparate regions poses intriguing questions concerning its biosynthesis and the formation and role of cell surface specialisations.

Reorganization of the endocytic compartment in regenerating liver

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocytes cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.

The Hepatocyte’s Plasma Membrane Domains. Interrelations with the Endocytic Compartment

The plasma membrane of the hepatocyte consists of three major functionally and biochemically characterised domains (Evans, 1980; 1981). The isolation of membrane vesicles or sheets from the sinusoidal, lateral and canalicular domains has allowed many aspects of cell surface physiology to be investigated, for example, the properties of transport mechanisms and details of the channels operational at the canalicular and sinusoidal poles of the cell have been identified (Berk et al., 1986; Meier,1986). The polarised organisation of the surface membrane of the hepatocyte raises basic questions regarding the nature of the sorting mechanisms directing newly synthesised proteins to different domains and the need to circumvent the barrier posed by tight junctions to lateral diffusion of proteins between the apical and baso-lateral domains (Evans et al., 1980; Bartles et al., 1988). The movement of ions, organic solutes, metabolites etc. from the blood to the bile across the interior of the hepatocyte raises questions as to the mechanisms, routes and the nature of the membrane networks involved.

Protein and antigen phosphorylation in the tegument of Schistosoma mansoni

Incubation of male and female Schistosoma mansoni for 3-6 h in media containing [32P]phosphate indicated that a large number of proteins extending across a wide molecular weight range were phosphorylated. The nature of the phosphorylated proteins was investigated by using three methods for removal of tegumental membranes. A 94 kDa polypeptide in the tegument became rapidly phosphorylated in vivo and it was also phosphorylated by incubation of isolated tegumental membranes in [32P]ATP in the presence of a protein kinase. After incubation of parasites in vivo for longer periods, other phosphorylated polypeptides of 62, 60, 57, 32 and 28 kDa were also identified. The major phosphorylated polypeptide immunoprecipitated from schistosomes by antisera raised in mice to irradiated cercariae was 62 kDa; others, of 48, 43 and 32 kDa, were also identified, using antisera raised in mice chronically infected with cercariae. The results suggest that mechanisms for receptor-mediated transmembrane signalling occur in the tegument of schistosomes.

Preparation of Synaptic Junctional Structures by Phase Partitioning in the Presence of n-Octyl-Glucoside

We have developed a simple and rapid method for the preparation of post-synaptic densities (PSD’s) and synaptic junctional complexes from mammalian brain. By morphological criteria the PSD preparation is pure, being essentially free of organised membrane and other contaminants and the yield is high (70 μg protein/g wet weight starting material). The method involves the use of the relatively mild neutral detergent, n-octyl-glucoside (OG) in conjunction with phase partitioning.