Human adipose tissue–derived mesenchymal stromal cells and their phagocytic capacity

Mesenchymal stromal cells (MSCs) have evidenced considerable therapeutic potential in numerous clinical fields, especially in tissue regeneration. The immunological characteristics of this cell population include the expression of Toll‐like receptors and mannose receptors, among others. The study objective was to determine whether MSCs have phagocytic capacity against different target particles. We isolated and characterized three human adipose tissue MSC (HAT‐MSC) lines from three patients and analysed their phagocytic capacity by flow cytometry, using fluorescent latex beads, and by transmission electron microscopy, using Escherichia coli, Staphylococcus aureus and Candida albicans as biological materials and latex beads as non‐biological material. The results demonstrate that HAT‐MSCs can phagocyte particles of different nature and size. The percentage of phagocytic cells ranged between 33.8% and 56.2% (mean of 44.37% ± 11.253) according to the cell line, and a high phagocytic index was observed. The high phagocytic capacity observed in MSCs, which have known regenerative potential, may offer an advance in the approach to certain local and systemic infections.     

Acceleration of α-Synuclein Aggregation by Exosomes

Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.     

Phospholipid Membrane-Mediated Hemozoin Formation: The Effects of Physical Properties and Evidence of Membrane Surrounding Hemozoin

Phospholipid membranes are thought to be one of the main inducers of hemozoin formation in Plasmodia and other blood-feeding parasites. The “membrane surrounding hemozoin” has been observed in infected cells but has not been observed in in vitro experiments. This study focused on observing the association of phospholipid membranes and synthetic β-hematin, which is chemically identical to hemozoin, and on a further exploration into the mechanism of phospholipid membrane-induced β-hematin formation. Our results showed that β-hematin formation was induced by phospholipids in the fluid phase but not in the gel phase. The ability of phospholipids to induce β-hematin formation was inversely correlated with gel-to-liquid phase transition temperatures, suggesting an essential insertion of heme into the hydrocarbon chains of the phospholipid membrane to form β-hematin. For this study, a cryogenic transmission electron microscope was used to achieve the first direct observation of the formation of a monolayer of phospholipid membrane surrounding β-hematin.     

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL''s mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL''s effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL''s binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.     

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents β-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ∼10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ∼30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.     

ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPγ-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.     

Phospholipid composition of a plasma membrane-enriched fraction from developing soybean roots

Phospholipid polar head group and fatty acid composition were determined for plasma membrane enriched fractions from developing soybean root (Glycine max [L.] Merr. cult. Wells II). Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots at pH 7.8 and in the presence of 5 millimolar ethylenediaminetetraacetate, 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N tetraacetic acid, and 10 millimolar NaF. Lipid extracts analyzed for phospholipid composition revealed two major phospholipid components: phosphatidylcholine and phosphatidylethanolmine. Minor phospholipid components identified were phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol. Lipid degradation by endogenous phospholipase D during membrane isolation at pH 6.5 and in the absence of chelating agents and NaF resulted in the recovery of large amounts of phosphatidic acid. Phosphatidylcholine was the principal substrate for phospholipase D.     

Coarsening Dynamics of Domains in Lipid Membranes

We investigate isothermal diffusion and growth of micron-scale liquid domains within membranes of free-floating giant unilamellar vesicles with diameters between 80 and 250 μm. Domains appear after a rapid temperature quench, when the membrane is cooled through a miscibility phase transition such that coexisting liquid phases form. In membranes quenched far from a miscibility critical point, circular domains nucleate and then progress within seconds to late stage coarsening in which domains grow via two mechanisms 1), collision and coalescence of liquid domains, and 2), Ostwald ripening. Both mechanisms are expected to yield the same growth exponent, α = 1/3, where domain radius grows as time^α. We measure α = 0.28 ± 0.05, in excellent agreement. In membranes close to a miscibility critical point, the two liquid phases in the membrane are bicontinuous. A quench near the critical composition results in rapid changes in morphology of elongated domains. In this case, we measure α = 0.50 ± 0.16, consistent with theory and simulation.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.     

ROLE OF THE SARCOPLASMIC RETICULUM IN GLYCOGEN METABOLISM : Binding of Phosphorylase, Phosphorylase Kinase, and Primer Complexes to the Sarcovesicles of Rabbit Skeletal Muscle

Sarcoplasmic vesicles and β-glycogen particles 30–40 mµ in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mµ, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10–20 mµ in diameter attached to the outer surface of the membranes. These particles disappear after α-amylase treatment. Incubation of the sarcovesicular fraction with ¹⁴C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.     

Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted β-muricholic acid almost completely into hyodeoxycholic acid.     

STUDIES ON SEEDS : IV. Lipid Composition of Bean Cotyledon Vesicles

Lipid content has been determined for two types of lipid-rich vesicles isolated from bush bean cotyledon at 24 hr of germination. The larger, nonassociating vesicles are four to six times richer in triglyceride than the smaller vesicles which associate strongly among themselves, as well as with smooth membranes in the cell. The larger vesicles contain about 640 µmoles of phospholipid per gram of protein, while the smaller vesicles have only one-half to two-thirds as much phospholipid per gram of protein. The ratio of individual phospholipids in both kinds of vesicles is close to 20% phosphatidylethanolamine, 60% phosphatidylcholine, and 20% phosphatidylinositol. The fatty acid composition of all phospholipids is similar, and quite different from that of triglyceride, which contains twice as much linolenic acid and less than one-fourth as much palmitic acid. Pea cotyledon has quantitatively the same lipid content as bean cotyledon.     

α-Synuclein Oligomers with Broken Helical Conformation Form Lipoprotein Nanoparticles

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as “discs.” Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7–10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.     

Gram-Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria

Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected. Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity.     

The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl

Extracellular components released from mycelia of the α and β races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible α race and compatible β race. Elicitor activity, however, was greater in the α race fractions. Elicitor purified from α race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.     

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Background & Aims

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

Methods

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

Results

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

Conclusions

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.     

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

The effects of heat shock on the synthesis of α-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25°C to 40°C for 3 hours, inhibits the accumulation of α-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca²⁺. When ER is isolated from heat-shocked aleurone layers, less newly synthesized α-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca²⁺ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.