High Strength Buffer Research Articles

Study of human erythrocyte membrane protein interactions by selective solubilization of Triton-skeletons

The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in presence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, protein 4.1 and actin present in erythrocyte skeletons does not depend on the molarity of NaCl used. In contrast ankyrin, protein band 3 and protein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protein interactions inside the skeleton. Solubilization was performed by Tris, a non-selective disruptive reagent, or by p-mercuribenzene sulfonic acid (PMBS), which principally release spectrin and actin. Tris action was assessed by calculation of the percentage of solubilized proteins, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With these two reagents, we observed a lower dissociation of skeletons prepared with high ionic strength buffer. Erythrocyte pretreatment with okadaic acid, an inhibitor of serine-threonine phosphatases, revealed a phosphorylation-induced skeleton gelation and a better resistance to Tris-solubilization.

Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries.

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins

A sample stacking procedure is presented for the capillary electrophoretic (CE) separation of paralytic shellfish poisoning (PSP) toxins dissolved in high ionic strength buffers. The application of such a stacking procedure prior to the zone electrophoretic separation is demonstrated for the analysis of decarbamoyl toxins arising from the digestion of PSP toxins by an hydrolytic enzyme from little neck clams ( Protothaca staminea). Improvements in separation efficiency facilitated identification and quantitation of substrates and enzymatic products present in the digest using CE. The separation conditions developed were found to be entirely compatible with electrospray mass spectrometry, which permitted the analysis of PSP toxins and their decarbamoyl derivatives present in the low micromolar range in crude enzyme digests. The products released during the enzymatic digestion were identified using CE combined with tandem mass spectrometry.

Similar DNA binding properties of free p70 (Ku) subunit and p70/p80 heterodimer

The Ku antigen consists of 70 and 80 kDa protein subunits (p70 and p80, respectively) that form the DNA binding component of a DNA-dependent protein kinase (DNA-PK). It is controversial whether the interaction of Ku with DNA is mediated by p70 alone or requires formation of p70/p80 dimers. In the present studies, the DNA binding properties of p70/p80 heterodimers and full-length human p70 expressed in the absence of p80 were investigated. The binding of free p70 and p70/p80 heterodimers to DNA showed similar sensitivity to high ionic strength buffers. Competitive DNA binding studies revealed that free p70, like the p70/p80 heterodimer, bound preferentially to linear double stranded DNA fragments, whereas tRNA and closed circular DNA molecules competed poorly with the radiolabeled linear DNA for binding to Ku. These studies suggest that free p70 and p70/p80 heterodimers have similar DNA binding properties, and that the interaction of Ku with DNA may depend primarily on the p70 subunit, possibly with implications for the assembly and function of DNA-PK.

A sandwich ELISA to detect VHSV and IPNV in turbot

The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.

A laccase‐like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues

SummaryStem tissues from different internodes of 4–6 week‐old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p‐diphenol:O2 oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H2O2 suggested that p‐diphenol:O2 oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase‐like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase‐like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall‐degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.

Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations.

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1h at 37°C to determine whether or not such an incubation might result in the redistribution of nuclear polypetides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37° C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37°C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Effects of negative charges of a model for bovine pancreatic trypsin inhibitor folding intermediate on the peptide folding.

A peptide model (called P alpha P beta) of bovine pancreatic trypsin inhibitor (BPTI) for studying the peptide folding structure was designed as a crucial folding intermediate. The P alpha P beta model folded but it was unstable at low pH. Four acidic groups in P alpha P beta: Glu-49, Asp-50, and C-termini at Phe-33 and Ala-58, were replaced individually with an Ala (in case of Glu and Asp) and an amide group (for C-terminal carboxyl groups) to study the effects of these negative charged groups on folding structure in terms of thermal stability and pH dependence. Substituting the Glu-49 or Asp-50 with Ala and blocking the C-terminus carboxyl group of Phe-33 in P beta destabilized the structure, but blocking the negative charge of C-terminus in Ala-58 of P alpha stabilized the structure at neutral pH. These results can be interpreted in terms of helix dipole momentum effects and/or salt bridge formation between Asp-50 and Arg-53, and of electrostatic interaction between positive and negative charges, which stabilized the structure. The melting temperature of model peptides (Tm) in low ionic strength buffer were lower than that in high ionic strength buffer except the C-terminus blocking of P alpha.

Assay of Tubulin Acetyltransferase Activity in Subcellular Tissue Fractions

An assay for α-tubulin acetyltransferase (TAT) activity based on affinity isolation of labeled acetylated α-tubulin was developed for use with crude subcellular fractions. Microtubules were polymerized and immobilized on an anti-α-tubulin-agarose and then incubated with the subcellular fraction and [3H]acetyl-coenzyme A (CoA). The labeled product was eluted from the antibody-agarose and the tritiated acetate incorporation determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot of the eluate verified the presence of acetyl-α-tubulin. Analysis of bovine retinal fractions showed the highest specific activity of tubulin acetyltransferase activity in the 27,000g pellet fraction (P2) of retinal homogenates. This transferase activity was proportional to the concentration of micro-tubule protein immobilized under polymerizing conditions and had an apparent Km of 3 μM for acetyl-CoA. The activity was solubilized from the P2 pellet by a high ionic strength buffer. The properties of the retinal TAT determined by this assay are very similar to those reported for a more purified enzyme preparation from Chlamydomonas flagella using the conventional trichlaroacetic acid precipitation assay and support the use of this method for samples in which high backgrounds prohibit use of the conventional assay.

Kurloff cell lysosomal arylsulphatases: presence of both cationic and highly anionic isoforms of the sole B class.

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or Lee-Vaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4-23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, 'classical' cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their Km (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO3 were also characteristic of this class of Asase. Finally, chondroitin-4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.

Conformational stability of the DNA-binding histone-like protein, HBsu, fromBacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W

From denaturation studies with urea a free energy delta GuH2O of unfolding of 49.8 kJ.mol-1 at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis. Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [theta] at 222 nm. For the calculation of delta Gu a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same delta Gu values. Four HBsu variants, each carrying one single point mutation ([F29W], [F47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The delta Gu values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F50W] and [F79W] in comparison to the wild type HBsu with delta delta Gu values of -9.2 kJ.mol-1, -7.5 kJ.mol-1, -5.9 kJ.mol-1, and -7.5 kJ.mol-1, respectively. Similar delta delta Gu values were obtained for the HBsu mutant proteins by thermal unfolding experiments.

Quantitation of Electrostatic and Hydrophobic Membrane Interactions by Equilibrium Dialysis and Reverse-Phase HPLC

Equilibrium dialysis and reverse-phase HPLC have been used for the sensitive and precise quantitation of both electrostatic and hydrophobic interactions of pep-tides and small molecules with lipid bilayers. We show that hydrophobic solutes are rapidly and quantitatively released from lipid dispersions when loaded onto a C4 reverse-phase HPLC column equilibrated in water + 0.1% trifluoroacetic acid and that the lipid molecules have no interfering effect on the chromatography. Peptides interacting electrostatically with bilayers are released quantitatively when a higher ionic strength buffer (water + 2% ammonium acetate) is used. As little as 50 ng of solute can be accurately quantitated even in the presence of milligram amounts of lipid. We demonstrate the application of these methods to the hydrophobic interactions between indoles and lipid bilayers and to the electrostatic interaction between defensins, which are cationic antibiotic peptides, and anionic bilayers. The high sensitivity allows nondestructive quantitation of submicrogram amounts of precious solutes and the high precision allows the heat capacity change, an important thermodynamic parameter, to be obtained from the partitioning data.

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Conductivity measurement as a convenient technique for determination of liposome capture volume

The reduction in conductivity seen between a buffer solution and a liposome preparation in that buffer was evaluated as a means of measuring liposome capture volume. Using DOPC and DOPG lipid to form negatively charged liposomes, conductivity measurements showed that conductivity of the liposome dispersion decreased as lipid concentrations of liposome preparations increased. Independent measurement of capture volumes by gel filtration chromatography showed that conductivity changes correlated with a liposome concentration dependent increase in capture volume. It is proposed that ions from the hydrating/suspending buffer normally contributing to conductivity were trapped within liposomes upon vesicle formation. These internalized and therefore shielded ions were not able to effectively contribute to conductivity of the liposome dispersion. For multilamellar vesicles (MLVs), capture volume was determined by reduction in conductivity over a large lipid concentration range and a broad buffer ionic strength range. Capture volume could also be determined for small unilamellar vesicles (SUVs). However, the greater number of exposed phospholipid head groups in high surface area SUVs contributed to conductivity of the liposome dispersion thereby limiting range of utility. A much higher ionic strength buffer (relative to MLVs) was required before conductivity of phospholipid no longer influenced conductivity of the dispersion. To expand this study, multilamellar vesicles having either neutral (DOPC) or positive (DOPC/stearylamine) charge were evaluated. Similar correlations were found between reduction in conductivity and mannitol entrapment (capture volume). These studies have confirmed that measurement of reduction in conductivity provides an easy and convenient method for determining liposome capture volume.

Absence of high levels of DNA polymerase α activity in the nuclear matrix prepared from mouse erythroleukemia cells

We have examined the association of DNA polymerase α activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase α activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37°C for 45 min prior to subfractionation. The recovery of DNA polymerase α activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH 4) 2 SO 4), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.

The Adsorption from Solutions of β-Lactoglobulin Mixed with Lactoferrin or Lysozyme onto Silica and Methylated Silica Surfaces

The adsorption from pure protein solutions and binary mixtures of proteins with opposite net charges was studied at pH 7 by in situ ellipsometry. The investigated proteins were β-lactoglobulin, lactoferrin, and lysozyme. On hydrophilic silica surfaces, β-lactoglobulin (negatively net charged) adsorbed in small amounts while lactoferrin and lysozyme (both positively net charged) adsorbed in higher quantities. All the proteins adsorbed readily to the methylated silica surfaces. On both surfaces the amounts of β-lactoglobulin adsorbed were higher and the ones for lactoferrin and lysozyme were lower in a high ionic strength buffer ( I = 0.17 M) compared to a buffer with low ionic strength ( I = 0.02 M). In mixtures of β-lactoglobulin and lactoferrin the proteins interact leading to high adsorbed amounts on the silica surface and to complete irreversibility with respect to rinsing with buffer. The effect is reduced by increasing the ionic strength of the buffer and by decreasing the overall concentration of proteins in the bulk. This is interpreted in terms of electrostatic interactions between the two proteins. The adsorbed amount from a β-lactoglobulin/lactoferrin mixture to a methylated silica surface is between the amounts for the pure proteins, and the adsorbate is a mixture of lactoferrin and β-lactoglobulin as indicated by measurements with radioactively labeled lactoferrin. An interesting observation was that the adsorbed amount from a β-lactoglobulin/lactoferrin mixture onto a methylated silica surface increased upon rinsing at high bulk concentrations and at low ionic strength. The adsorption from mixtures of β-lactoglobulin and lysozyme is also strongly influenced by electrostatic interactions. At low ionic strength a bulk precipitation takes place, which probably is the reason for the very high amounts adsorbed onto both surfaces examined. This effect is only partially reduced at higher ionic strength.

Chiral separation of β-blockers by high-performance capillary electrophoresis based on non-immobilized cellulase as enantioselective protein

Optical isomers of some basic parmaceutical drugs (β-blockers) were separated by means of high-performance capillary electrophoresis in a carrier-free solution, using the chiral recognition properties of a cellulase (cellobiohydrolase I). High resolution of the isomers and peaks with satisfactory symmetry were obtained only when the enzyme was dissolved at a high concentration (40 mg/ml; total 10 μg) in a buffer of high ionic strength (0.4 M sodium phosphate) supplemented with 2-propanol. Surprisingly, the isomer selectivity was lost when the electrophoresis was carried out in the buffers used for chromatographic separation of the isomers on a bed derivatized with cellulase. At pH 5.1 (the experimental pH), the enantiomers are positively charged and the enzyme is negatively charged. With the cathode at the detection end of the capillary the enzyme accordingly migrated away from the detection point and the enantiomers toward it. Disturbances in the UV detection of the enantiomers otherwise caused by the presence of the enzyme were thus avoided. As the runs are performed in the absence of a supporting medium the analyses can be automated easily, which also facilitates the screening of different proteins for their chiral recognition properties and studies to establish the optimum experimental conditions.

Characterization of wall‐bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza‐forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall‐bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall‐bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall‐bound isoform of acid invertase were found that function as β‐d‐fructofuranoside‐fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS‐PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin‐labeled protein. The Km values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase Km= 16 mM, tightly bound invertase Km= 8.6 mM). Activity of both wall‐bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH‐optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall‐bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.

Detection of Low Molecular Weight GTP-binding Proteins Associated with Rat Atrial Secretory Granules

Recent studies suggest that low molecular weight (LMW) GTP-binding proteins are involved in the vesicular transport of proteins along the secretory pathway. In order to determine whether LMW GTP-binding proteins are potentially involved in the regulated secretion of atrial natriuretic factor (ANF) from the atrial myocyte, rat atrial secretory granules were purified and the LMW GTP-binding proteins associated with these granules were detected in [α- 32P]GTP-blotting experiments. Three LMW GTP-binding proteins, displaying molecular weights of 23 000, 25 000 and 29 000, were usually observed in atrial secretory granule preparations. The 29 kDa GTP-binding protein (G 29), however, was typically the predominantly labelled band. the binding of [α- 32P]GTP to G 29 was guanine nucleotide specific and magnesium-dependent. Trypsin treatment of intact secretory granules markedly reduced the binding of [α- 32P]GTP to G 29. G 29 is tightly associated with the granule membrane, as evidenced by its resistance to solubilization by high ionic strength buffers but its sensitivity to solubilization with sodium cholate. When secretory granule preparations of rat anterior pituitary glands were examined for the presence of LMW GTP-binding proteins, a different pattern of LMW GTP-binding proteins was observed which did not include a predominantly labelled 29 kDa protein. The results of this study indicate that: (1) LMW GTP-binding proteins are present on atrial secretory granules and may therefore be involved in the regulated secretion of ANF and (2) tissue-specific differences exist between the LMW GTP-binding proteins associated with secretory granules.