Intracellular transport, organelle biogenesis and establishment of Golgi identity: impact of brefeldin A on the activity of lipid synthesizing enzymes.

1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phosphoglycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated. 2. In the presence of 5-10 micrograms/ml BFA, the incorporation of [3H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45-60%, and the production of endoplasmic reticulum transport vesicles was reduced 30-50%. 3. In Golgi membranes, the presence of 5-10 micrograms/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30-40%. 4. Addition of 5-10 micrograms/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine. 5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

Membrane biogenesis in the presence of ethanol.

To investigate the effect of ethanol on the intracellular transport in gastric epithelial cells, the in vitro system, generating transport vesicles which transfer mucus glycoprotein apopeptide (apomucin) from rough endoplasmic reticulum (RER) to Golgi, was assembled. The vesicles, generated from gastric mucous cell RER microsomes and labeled with [3H]palmitic acid, were isolated from the maternal RER and characterized. The electron microscopy revealed that these RER products consisted of 80 to 100 nm smooth membrane vesicles, while the immunochemical analyses showed that they contain apomucin but were devoid of the characteristic integral proteins of the RER membrane. Incubation of apomucin transporting vesicles with Golgi in the presence of UDP-[3H]galactose resulted in the formation of glycosylated mucin and fusion of the vesicles with Golgi. Formation of ER transport vesicles was dependent on the supply of lipid precursors, and the activity of phosphoglyceride and sphingolipid synthesizing enzymes. In the presence of 60 and 120 mM ethanol, the vesicles were formed, but their lipid composition was modified. The results suggest that ethanol-induced membrane alterations are initiated at the early stages of the membrane biogenesis and are first reflected in the lipid composition of the intracellular transport vesicles.

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.

Function of intracellular phospholipase A2 in vectorial transport of apoproteins from ER to Golgi.

1. The cytosolic fraction required in in vitro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A2 (PLA2) which assists ER vesicles fusion with Golgi. 3. The ability of the 76 kDa PLA2 to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained following dephosphorylation. 4. The data provide evidence that 76 kDa intracellular PLA2 is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.

Biogenesis of endoplasmic reticulum transport vesicles transferring gastric apomucin from ER to Golgi.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de novo synthesized phosphatidylcholine (PC) was incorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 mM N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Zn2+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed.

Mucin complexes: characterization of the "link" component of submandibular mucus glycoprotein.

1. Analysis of the submandibular saliva revealed that the secretion consists of mucin complexed with 150 kDa fibronectin fragment and DNA. 2. The kallikreins, secreted by the submandibular gland, appear to be responsible for the fibronectin fragmentation, since an identical peptide was also generated when fibronectin was subjected to incubation with the submandibular saliva or the purified enzyme. 3. The results provide evidence that the 150 kDa glycopeptide so-called salivary mucin "link" component is neither an integral part of the mucin molecule, nor linked to mucin subunits by disulfide bonds, but is a fibronectin fragment which associates with mucin. 4. Using mucin monoclonal antibody (3G12), it was revealed that the nonglycosylated (naked) 8-12 kDa fragment of the mucin molecule is responsible for the interaction of mucin with other components of saliva. 5. Under physiological conditions, the interaction of mucin with fibronectin on the luminal surfaces may be relevant in building mucous barrier and protection of the delicate oral epithelium from damage.

Purification of protein fatty acyltransferase and determination of its distribution and topology.

Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.

Hyperthermia, unlike ionizing radiation and chemical oxidative stress, does not stimulate proteolysis in erythrocytes.

1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. Gamma-irradiation of erythrocytes in the range of 50-1000 Gy also resulted in the induction of proteolysis. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39-49 degrees C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45 degrees C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.

Nature of the enhancement of the protective qualities of gastric mucus by sucralfate.

The effects of intragastric administration of an antiulcer drug, sucralfate, on the content, composition and physical properties of the mucus component of gastric mucosal barrier were investigated. The experiments were conducted with two groups of rats in which one group received twice daily for five consecutive days a dose of 100 mg sucralfate, while the control group received daily doses of saline. The animals were sacrificed 16 h after the last dose, their stomachs dissected and the mucosa subjected to measurements of the adherent mucus gel dimension and mucin content, evaluation of its viscosity, H+ retardation capacity and hydrophobicity, and analysis of its components and molecular form distribution of elaborated mucin. The results revealed that sucralfate elicited only a minor (8%) increase in mucus gel dimension, while sulfo- and sialomucin contents of the gel increased by 63 and 81%, respectively. The changes in mucus gel mucin content with sucralfate were accompanied by a slight (9.5%) increase in mucus H+ retardation capacity, 1.9-fold increase in viscosity, and a 60% increase in the gel hydrophobicity. Chemical analyses indicated that the mucus elaborated in the presence of sucralfate exhibited 14% lower protein content and 62% higher content of carbohydrate than that of control, displayed similar levels of total lipids and covalently bound fatty acids, but its neutral lipid content was significantly higher. Furthermore, the gastric mucus of the sucralfate group showed a marked increase in the proportion of the high-molecular-weight form of mucin, while the low-molecular-weight form predominated in the control group. The results indicate that the protective qualities of sucralfate lie mainly in its ability to enhance the gastric mucus gel viscosity, hydrophobicity, content of mucin and its proportion in the high-molecular-weight form.

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance.

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.

Effect of cell age on the quenching of erythrocyte membrane protein fluorescence.

The accessibility of membrane protein tyrosyl and tryptophanyl residues for fluorescence quenchers (acrylamide and iodide) was compared in membranes isolated from various age fractions of bovine erythrocytes. An increase in the quenching efficiency of membrane protein fluorescence was found with advancing erythrocyte age, as demonstrated by elevated values of the Stern-Volmer constant. These results provide evidence for a progressive change in membrane protein organization during erythrocyte aging, enhancing the availability of membrane proteins for interactions with external factors.

Ligand and lipid domain stabilization of a membraneous Ca2+-ATPase during hyperthermia.

The susceptibility of the membranous Ca2+-ATPase of sarcoplasmic reticulum to enzymatic inactivation at hyperthermic temperatures was investigated. Inactivation produced a break in the Arrhenius plot at 45-46 degrees C and was accompanied by an increased mobility of spin label, covalently attached to the Ca2+-ATPase. MgADP and MgATP exerted a markedly stabilizing effect on inactivation, both at pH 7.0 and in acidic media. By contrast, high-affinity Ca2+ or Mg2+ binding only moderately stabilized Ca2+-ATPase (inactivation rates were decreased 2-3 times), and this effect was non-additive, i.e., only observed in the absence of the other divalent cation. But withdrawal of K+ and Na+ gave rise to a pronounced destabilization that could be reversed efficiently by high concentrations of Ca2+ or Mg2+. These results are compared with a previous study on detergent solubilized Ca2+-ATPase (Møller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920) which showed the enzyme to be markedly stabilized by Ca2+ as well as by nucleotide. It is concluded that, due to the presence of nucleotide, inactivation of Ca2+-ATPase is not likely to occur during malignant hyperthermia and that the native environment of the lipid bilayer provides stabilization of the membrane-embedded and Ca2+-translocating domain of the Ca2+-ATPase.

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis.

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.

Aged erythrocytes exhibit decreased anion exchange.

The rate of transport of [32P] phosphate into human and bovine erythrocytes and of a spin-label analogue of phosphate (Tempo-phosphate) into human erythrocytes was found to decrease with increasing erythrocyte age by 15-20% when comparing 20% most dense cells with 20% of lightest cells. The activation energy of Tempo-phosphate transport did not show significant changes upon erythrocyte aging.

An assay for proteolytic activity using spin-labeled substrates.

A method is proposed for the assay of proteolytic activity based on the measurement of changes in the electron spin resonance spectra (increase in the ratio of weakly to strongly immobilized spin label residues) of substrate proteins labeled with a maleimide nitroxide derivative.

Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes.

Decrease in electrophoretic mobility of erythrocytes, increase in Km of erythrocyte membrane acetylcholinesterase and decrease in the binding constant of 8-anilino-1-naphthalene sulfonate to erythrocyte membranes demonstrate a decrease in surface charge density of bovine erythrocytes during in vivo aging. This phenomenon seems to be species-specific; it may be due to a diminution of the sialic acid content but may also be contributed by conformational changes of membrane proteins.

Hyperthermic modification of bleomycin--DNA interaction detected by electron spin resonance.

Electron spin resonance spectra of DNA labeled with each of four spin-labeling compounds have been studied to detect interaction between the antibiotic bleomycin and DNA. Only one of these labels, compound IV, resulted in a modified spectrum when bound to DNA and the latter was subjected to bleomycin. This property has been used to monitor DNA-bleomycin interactions under physiological and hyperthermic conditions. Bleomycin produced an increase in rotational correlation time of the residue bound to DNA at 37 degrees C and a significantly higher increase at 43 degrees C. Some effect was still detected with bleomycin at 37 degrees C after preheating at 43 degrees C. Parallel studies have revealed enhanced binding of 59Fe-bleomycin to DNA during and after hyperthermic treatment.

Aging of the erythrocyte. XVIII. Changes in kinetic properties of acetylcholinesterase.

Comparison of kinetic properties of acetylcholinesterase in membrane preparations from various density (age) fractions of bovine erythrocytes revealed in increase in Km value, ascribed to decrease in membrane surface potential, with increasing cell age. Activation energy of the enzyme decreased during red cell aging.

Aging of the erythrocyte--XVII. Changes in the properties of superoxide dismutase.

1. An increase in the sensitivity of superoxide dismutase (SOD) to the action of diethyldithio-carbamate and cyanide was observed in red cell fractions of greater age. 2. It suggests that SOD inactivation during erythrocyte aging is not an "all or none" process but involves transition(s) between enzyme forms of different properties. 3. An increase in the ratio of less mobile to the more mobile SOD bands was observed by polyacrylamide-gel-electrophoresis in older erythrocytes.