Labeling of membranes from erythrocytes and corn with fluorescamine

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.     

Characterization of ATPase activity associated with corn leaf plasma membranes

A Mg²⁺-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg²⁺-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K_m of 0.28 millimolar Mg²⁺-ATP was determined for the K⁺-ATPase, and the principal effect of potassium was on the V_max for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.     

The isolation, culture and regeneration of Petunia leaf protoplasts

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

The isolation of leaf protoplasts from the submerged aquatic angiosperm Potamogeton lucens L.

Abstract. A method for isolating protoplasts from leaves of the submerged aquatic angiosperm Potamogeton lucens L. is described. The protoplasts are produced enzymatically from leaf strips using 1.5% (w/v) Cellulysin, 0.3% (w/v) Macerozyme R10, and 2.5% (v/v) β-glucuronidase at 27°C in the dark. Subsequently the protoplasts are purified on a discontinuous Ficoll gradient. The yield obtained is approximately 20% of the starting material on a chlorophyll basis. The viability is high, namely more than 90% as estimated with Evans Blue. Cells of the intact leaves of P. lucens can use HCO₃⁻ for photosynthesis. ¹⁴CO₂ fixation experiments at pH 6.0 and 8.5 suggest that these isolated protoplasts can also use bicarbonate as a carbon source.     

A comparative spin-label study of isolated plasma membranes and plasma membranes of whole cells and protoplasts from cold-hardened and nonhardened winter rye

Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between −40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used.     

Properties of guaiacol peroxidase activities isolated from corn root plasma membranes

Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, this study is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highly enriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightly bound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography (pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, and the corresponding protein bands were identified by heme staining. The activities of the peroxidase enzymes responded different to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Because the enzymes were localized at the PM and were not effected by p-chloromercuribenzoate, they were probably class III peroxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular mass of 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractions by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with heme staining confirmed the estimated molecular masses of the size exclusion chromatography.     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Labeling of glucagon binding components in hepatocyte plasma membranes.

A photosensitive derivative of glucagon, ¹²⁵I-N^?-4-azido-2-nitrophenyl-glucagon, has been synthesized and used to specifically label glucagon binding proteins in hepatocyte plasma membranes. Photolysis of the derivative in the presence of a membrane suspension results in the incorporation of radioactivity primarily into membrane components with a molecular weight range of 23,000–25,000. The binding properties of the derivative are essentially identical to that observed for glucagon. The binding of ¹²⁵I-NAP-glucagon was completely inhibited in the presence of glucagon (3 μM) while greater than 90% of the covalent labeling was also inhibited in the presence of glucagon. These studies suggest that the labeled membrane protein may be a component of the glucagon receptor.     

Nonselective currents and channels in plasma membranes of protoplasts from coats of developing seeds of bean

In developing bean (Phaseolus vulgaris) seeds, phloem-imported nutrients move in the symplast from sieve elements to the ground parenchyma cells where they are transported across the plasma membrane into the seed apoplast. To study the mechanisms underlying this transport, channel currents in ground parenchyma protoplasts were characterized using patch clamp. A fast-activating outward current was found in all protoplasts, whereas a slowly activating outward current was observed in approximately 25% of protoplasts. The two currents had low selectivity for univalent cations, but the slow current was more selective for K(+) over Cl(-) (P(K):P(Cl) = 3.6-4.2) than the fast current (P(K):P(Cl) = 1.8-2.5) and also displayed Ca(2+) selectivity. The slow current was blocked by Ba(2+), whereas both currents were blocked by Gd(3+) and La(3+). Efflux of K(+) from seed coat halves was inhibited 25% by Gd(3+) and La(3+) but was stimulated by Ba(2+) and Cs(+), suggesting that only the fast current may be a component in the pathway for K(+) release. An "instantaneous" inward current observed in all protoplasts exhibited similar pharmacology and permeability for univalent cations to the fast outward current. In outside-out patches, two classes of depolarization-activated cation-selective channels were observed: one slowly activating of low conductance (determined from nonstationary noise to be 2.4 pS) and another with conductances 10-fold higher. Both channels occurred at high density. The higher conductance channel in 10 mM KCl had P(K):P(Cl) = 2.8. Such nonselective channels in the seed coat ground parenchyma cell could function to allow some of the efflux of phloem-imported univalent ions into the seed apoplast.     

A method for isolation of protoplasts from dermatophytes

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.     

Structural characterization of two aquaporins isolated from native spinach leaf plasma membranes

Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.     

Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2--lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.     

Standardized isolation of protoplasts

Isolation of leaf mesophyll protoplasts from tobacco (Nicotiana tabacum) is facilitated using a specially designed digestion chamber. The chamber's airtight seals and various ports reduce the complexity, time and contamination risks which may be associated with standard isolations. The polished glass viewing window allows continuous monitoring of protoplast release, thus facilitating more precise determinations of the optimal digestion time. In concert with a simplified centrifugation step, the protoplast isolation procedure is greatly standardized.     

A new method for the isolation of plasma membranes from amphibian embryos

Summary A method for the isolation of plasma membranes is described. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), a heterobifunctional reagent, is covalently linked to protein amino groups in plasma membranes of intact cells. After homogenization of the cells the plasma membranes can be separated from other cell components by selective coupling to reduced Thiopropyl-Sepharose 6B and then recovered after treatment with 2-mercaptoethanol.