Albumin secreted by rat liver bypasses Golgi apparatus cisternae.

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [3H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60-90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elements of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.

Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicle and Golgi apparatus fractions of rat liver.

Coated vesicles were isolated from rat liver in about 80% fraction purity as determined from electron microscopy and analyses of marker enzymes and compared with Golgi apparatus and other membrane fractions isolated in parallel. The fractions were enriched in NADH-monodehydroascorbate reductase, ascorbate oxidase and ascorbic acid. The NADH-monodehydroascorbate reductase and ascorbate oxidase of the Golgi apparatus and coated vesicles differed from that of the endoplasmic reticulum in being inhibited by the sodium selective ionophore, monensin, at physiological concentrations while these activities were stimulated by ethylenediaminetetraacetic acid in coated vesicles but not in Golgi apparatus. Activities of both coated vesicles and Golgi apparatus fractions depleted in the coat protein, clathrin, were activated by the addition of clathrin-rich supernatant fractions. The results are discussed in the context of monodehydroascorbate as an acceptor for electron transport-mediated transfer of electrons from NADH by coated vesicles as part of a possible mechanism to drive membrane translocations or to acidify the interiors of vesicles.

Immunocytochemical localization of class I H-2 histocompatibility antigens of mouse liver.

The subcellular location of class I H-2 histocompatibility antigens was determined for mouse liver using immunocytochemical techniques and correlated with information determined by cell fractionation and analysis in situ. Surface antigens first were localized by standard procedures involving surface labeling with ferritin-labeled antibody. This approach could not be used for internal membranes either in situ or in fractions since the antigens are not expressed at the cytoplasmic surface. For this purpose, thin sections of tissues embedded in Lowicryl were analyzed and quantitated. The in situ analysis confirmed the presence of H-2 antigens on internal membrane compartments as well as on the cell surface and helped rule out the possibility that distributions based on analyses by immunoprecipitation of fractions of internal membranes were influenced greatly by plasma membrane contamination. Quantitation was provided by immunoprecipitation of H-2 antigens from radioiodinated or metabolically labeled isolated and highly purified cell fractions. The findings establish the presence of class I H-2 histocompatibility antigens in endoplasmic reticulum, Golgi apparatus and plasma membrane in the approximate ratios of 1:3:7. No class I H-2 histocompatibility antigens could be detected in mitochondria, salt extracts of isolated membranes or NP-40-insoluble membrane material.

A specific radioimmunoassay for the measurement of gadoteridol, a contrast agent for magnetic resonance imaging in biological fluids.

Gadoteridol, a nonionic gadolinium chelate, is currently being evaluated for contrast-enhanced magnetic resonance imaging. A radioimmunoassay (RIA) has been developed for the measurement of gadoteridol in biological fluids. The RIA has a range of 0 to 25 micrograms/mL and has the sensitivity to detect 0.05 microgram/mL of gadoteridol. Satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved with 12.5% polyethylene glycol. A quantitative recovery of the exogenous analyte was obtained at all concentrations of gadoteridol tested. Linearity in both serum and urine was satisfactory. Intraassay coefficients of variation were 6.4 and 2.8% for the low and medium controls, respectively. Interassay coefficients of variation were 5.4, 3.8, and 12.2% for the low, medium, and high controls, respectively. Cross reactivities of the ligand 5 and the calcium salt 6 were 37 and 29%, respectively. Clinical samples from the ascending dosage studies were analyzed by the gadoteridol RIA. The results obtained from the serum specimens demonstrated an excellent linear proportionality between drug concentration in blood and administered dosage of gadoteridol. Cumulative urinary excretion data showed that 94% of the drug was excreted in the urine within 24 h.

Isolation of plasma membrane, golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver.

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2-3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K+-stimulated p-nitrophenyl phosphatase and (Na+K+) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.

Flow kinetics of mouse histocompatibility antigens.

Major histocompatibility antigens of the mouse (H-2 antigens) are found on a variety of different cell types and constitute a class of integral membrane-bound glycoproteins involved in tissue graft rejection and immune surveillance. We used monospecific alloantibodies directed against mouse H-2 antigens and standard pulse-chase technique to investigate the flow kinetics of delivery of newly synthesized membrane constituents to the cell surface. [35S]Methionine was injected intraperitoneally, and, after a chase with unlabeled methionine, livers were excised and fractionated into endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions. Label first appeared in H-2 antigens located within the endoplasmic reticulum. Maximum specific activity observed between 5-7 min after injection of label was followed by a rapid loss of label. H-2 antigens of Golgi apparatus also were labeled early. Peak specific activity observed 15-25 min after injection of label was again followed by rapid loss of label. H-2 antigens of the plasma membrane were labeled last and appeared to accumulate radioactivity with no evidence of rapid turnover. These results provide evidence for a precursor-product relationship between H-2 antigens located within the cell on membranes of endoplasmic reticulum and Golgi apparatus and those on the plasma membrane. Flow of individual membrane-bound glycoproteins from their sites of synthesis and insertion into the membrane of the endoplasmic reticulum to the plasma membrane through the Golgi apparatus is indicated.