Theo P. van der Krift

Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the non-specific lipid transfer protein (sterol carrier protein 2)

An antibody against the non-specific lipid transfer protein from rat liver was purified by immunoabsorbent affinity chromatography. This antibody in conjunction with protein A-colloidal gold was used to localize the transfer protein in rat liver by electron microscopy. Labeling by this immunocytochemical technique was found to be mainly restricted to the peroxisomes; low labeling was observed in the cytoplasm. Subsequent analysis of isolated peroxisomes by immunoblotting indicated that the non-specific lipid transfer protein (mol. wt. 14800) was absent from this organelle and that a protein of molecular weight 58000 was responsible for the immunological response. Immunoblotting of the membrane-free cytosol showed the presence of both proteins. It remains to be established to what extent the non-specific lipid transfer protein in the cytosol and the high-molecular weight protein in the peroxisomes are related.

Determination of nonspecific lipid transfer protein in rat tissues and Morris hepatomas by enzyme immunoassay.

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2-2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5-10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.

PHOSPHOLIPID TRANSFER ACTIVITIES IN MORRIS HEPATOMAS AND THE SPECIFIC CONTRIBUTION OF THE PHOSPHATIDYLCHOLINE EXCHANGE PROTEIN

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 9633 and 7777, and compared to the activities found in normal and host liver. A 2-3-fold increase was found in the phosphatidylcholine and phosphatidylinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.

Tissue distribution and subcellular localization of phosphatidylcholine transfer protein in rats as determined by radioimmunoassay.

A radioimmunoassay for the phosphatidylcholine-transfer protein from rat liver was used to measure levels of PC-transfer protein in rat tissues. The assay as described before (Teerlink, T., Poorthuis, B.J.H.M., Van der Krift, T.P. and Wirtz, K.W.A., Biochim. Biophys. Acta 665 (1981) 74-80) was modified in order to measure PC-transfer protein in tissue homogenates and subcellular membrane fractions. To this end both a detergent (Triton X-100) and a proteolytic enzyme inhibitor (aprotinin) were added to the assay medium. The radioimmunoassay measured levels of PC-transfer protein in the range of 5-50 ng and was specific for PC-transfer protein from rat tissues. Subcellular distribution studies showed that in 10% (w/v) homogenates of liver approximately 60% of the PC-transfer protein was present in the 105000 X g supernatant fraction, the remainder being evenly distributed over the particulate fractions. PC-transfer protein associated with the particulate fractions was almost completely removed by a single washing step, suggesting a dynamic equilibrium between membrane-bound and soluble PC-transfer protein. Both 105000 X g supernatants and homogenates of various rat tissues were assayed. The highest levels of PC-transfer protein were measured in liver and intestinal mucosa. Lower values were found in kidney, spleen and lung, whereas heart and brain contained hardly any PC-transfer protein. PC-transfer protein levels in regenerating rat liver did not differ significantly from levels in normal liver. In fetal lung a change in PC-transfer protein content during development was observed, with a clear maximum 2 days before term, suggesting an involvement of PC-transfer protein in the secretion of lung surfactant.

Visualization of contact sites between outer and inner envelope membranes in isolated chloroplasts

Abstract Contact sites between outer and inner envelope membranes of isolated chloroplasts have been studied at the ultrastructural level by two different electron microscopical methods, i.e., freeze fracturing and freeze substitution followed by ultrathin sectioning. Both methods demonstrate that approx. 10% of the chloroplast population exhibits blister-like structures. Cross fractures and ultrathin sections of freeze-substituted chloroplasts reveal that the blisters originated from a separation of the outer and inner envelope membranes. Exposure of isolated chloroplasts to hypertonic conditions results in an almost complete separation of the two envelope membranes, except for small regions in which the two membranes are in close contact. These contact sites are clearly recognized in freeze-fracture replicas by ridges of a high density of intramembrane particles. In addition, cross fractures and thin sections of freeze-substituted chloroplasts demonstrate the presence of small vesicles associated with the outer envelope membrane. As indicated by the opacity of the vesicle and demonstrated by immuno-gold labeling, these vesicles, which originate from the inner envelope membrane, contain stroma-derived proteins.

A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno-electron microscopical studies

A combination of cryofixation without pre‐treatment, freeze‐substitution and low‐temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P. chrysogenum colonies were grown between dissected‐dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze‐substitution media, but it was reduced after embedding in Epon 812.

The taxonomic position of Asterodon, Asterostroma and Coltricia inferred from the septal pore cap ultrastructure

The ultrastructure of the septal pore cap (SPC) of Asterodon, Asterostroma and Coltricia were examined to establish the taxonomic position of these genera. Asterostroma has dolipores with perforate SPCs and is classified in the Lachnocladiaceae. In contrast, Asterodon and Coltricia have dolipores with imperforate SPCs and belong to the Hymenochaetaceae. Other selected species of genera belonging to the Hymenochaetaceae like Hydnochaete, Coltriciella, Inonotus, Onnia, and Cyclomyces also contained imperforate SPCs. Coltriciella, Inonotus and Cyclomyces moreover presented a lamella of endoplasmic reticulum above the imperforate SPC after chemical fixation. Such a lamella could rarely be observed in Coltricia only after high-pressure freezing and freeze substitution. Cryofixed fungal cells of Cyclomyces and Coltricia showed dierences in the architecture of the matrix of the SPC. Coltricia showed a more layered matrix structure than the SPC of Cyclomyces. In addition, transmission- and scanning electron microscopy revealed an indent in the centre of the imperforate SPC of Cyclomyces, indicating a reduced thickness, and resulting into a tented profile in crosssections.

Measurement of phosphatidylcholine transfer protein in rat liver and hepatomas by radioimmunoassay

An antiserum was raised against the phosphatidylcholine transfer protein from rat liver by immunization of rabbits. The antiserum was shown to be specific for this protein. A double-antibody radioimmunoassay for the phosphatidylcholine transfer protein was developed. In order to economize the use of second antibody (immunobeads), the specific anti-phosphatidylcholine transfer protein-IgG fraction isolated by affinity chromatography was used. Phosphatidylcholine transfer protein was labelled with 125I by the glucose oxidase-lactoperoxidase method and purified from the reaction mixture by affinity chromatography. Approx. 80% of the tracer was immunoprecipitable. The operating range of the assay was from 4 to 50 ng of transfer protein. This assay was used to determine the levels of phosphatidylcholine transfer protein in the 105000 x g supernatant fractions of rat liver and Morris hepatomas 7777, 7787 and 9633. The values obtained for the tumors were in good agreement with results previously obtained by immunotitration of the phosphatidylcholine transfer activity (Poorthuis, B.J.H.M., Van der Krift, T.P., Teerlink, T., Akeroyd, R., Hostetler, K.Y. and Wirtz, K.W.A., Biochim. Biophys. Acta 600 (1980) 376--386). For normal and host liver, the values determined by the radioimmunoassay were 2--4-fold higher.