High Strength Buffer Research Articles

Sarcoplasmic and myofibrillar proteins in white trunk muscle of salmon ( Salmo salar) after estradiol treatment

1. 1. Proteins from skeletal muscle of Atlantic salmon ( Salmo salar) were separated into two fractions, sarcoplasmic and myofibrillar, the sarcoplasmic proteins amounting to 47%. 2. 2. The actomyosin linkage of the myofibrillar proteins was broken by adding ATP MgCl 2 and myosin was brought into solution in high ionic strength buffer. 3. 3. The method was applied to fish treated with 17-β estradiol. The sarcoplasmic protein content per g wet wt or per mg of DNA was unchanged. The myofibrillar proteins decreased by 17%. Myosin heavy chain decreased to a simila extent as the total myofibrillar proteins. 4. 4. The advantage of the methods described is to relate changes in muscle protein metabolism to specific physiological functions of the tissue.

Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro: Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.

DNA hairpin loops in solution. Correlation between primary structure, thermostability and reactivity with single-strand-specific nuclease from mung bean

Hairpin structures formed by seven DNA inverted repeats have been studied by PAGE, UV(CD)-spectroscopy and nuclease cleavage. The hairpins consisted of (CG)3 stems and loops of 2, 3 and 4 residues. Thermal stabilities (Tm) have been determined in low and high ionic strength buffers, where the hairpins were structured in the B- and Z-DNA form respectively. The thermodynamic parameters of hairpin formation have been obtained by a two-state analysis of the hairpin-coil transitions. It is found that, on increasing the number of bases in the loop from 2 to 3 and 4, the Tms of the B-hairpins decrease, whereas the Tms of the same hairpins in the Z-form increase. This confirms previous evidence (1,2) that in a hairpin molecule the size and structure of the loop are modulated by the conformation of the helical stem. Moreover, B-hairpins with loops comprising 2, 3 and 4 bases have been digested with the single-strand-specific nuclease from mung bean. In our experimental conditions (0 degrees C) the nuclease preferentially cleaves the unbonded nucleotides of the loops. However, the rates of loop hydrolysis, which roughly follow a first-order kinetics, markedly depend on the size of the loop. At a ratio of 3 enzyme units/micrograms DNA, the half-lives of hairpins which are expected to form loops of 4, 3 and 2 residues are 90, 145 and 440 minutes respectively. Thermostability and enzymatic digestion data suggest that two-membered loops can be formed in B-hairpins but not in Z-hairpins.

Partial purification of human prostatic 5α-reductase (3-oxo-5α-steroid: NADP + 4-ene-oxido-reductase; EC 1.3.1.22) in a stable and active form

Human hyperplastic prostate tissue was homogenised in high ionic strength buffer and the post nuclear homogenate was incubated with 0.8% octyl glucoside and bovine brain lipids. Dialysis of the resulting liposome suspension yielded a preparation in which 5α-reductase was active and stable for at least three weeks and showed an increase in specific activity ( V max±SD=48.9±7.4pmol DHT/mg protein/ml) over that of the starting homogenate ( V max±SD=5.6±1.5pmol DHT/mg protein/min) of 8.7 times.

Muscle protein changes following eccentric exercise in humans

This study characterized changes in the protein composition of human muscle tissue after eccentric exercise. Four subjects performed 70 maximum eccentric, isokinetic actions of the forearm flexors with one arm. The other arm served as control. A biopsy of the biceps muscle of each arm was taken 2 days after exercise when muscles were very sore (mean = 8.0; 1 = normal; 10 = very, very sore), and muscle damage was documented by a mean decrease of 0.2 radians in the relaxed elbow angle. Proteins from the biopsy tissue were solubilized in a high ionic strength buffer containing several proteolytic inhibitors. Protein concentrations of the extracts were determined and identical amounts loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels (7.5, 12.5, and 17.5%). Densitometric analysis of the Coomassie brilliant blue stained gels revealed alterations in the amounts of three protein bands in the exercised tissue relative to the control. These changes were in the linear portion of the graph of absorbance versus protein amount. Wilcoxon's signed rank test showed the first two of the following bands to increase significantly in amount (P less than 0.062). The average percentage changes [mean (SEM)] for these bands were 63 (21), 39 (5), and 82 (35). The corresponding molecular weights determined from known standards were 76300 (860), 33200 (310), and 12000 (80) daltons, respectively. These changes imply that the increased synthesis, decreased degradation, or some combination thereof, of these three proteins may be necessary for the repair or regeneration response to exercise-induced muscle damage.

TFIIIA induced DNA bending: effect of low ionic strength electrophoresis buffer conditions

We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of the Xenopus somatic-type gene [Schroth, G.P. et al. (1989) Nature 340, 487-488]. In addition, we show that TFIIIA induced DNA bending is dramatically affected by the ionic conditions used during gel electrophoresis. By modifying the conditions of the electrophoresis, we can detect two distinct conformations for the TFIIIA/DNA complex. In very low ionic strength buffers, the degree of DNA bending in the complex is estimated to be about 25 to 30 degrees, whereas in higher ionic strength buffers it is about 60 to 65 degrees. These data explain the apparent discrepancy between our results and the results of another study in which it was claimed that TFIIIA did not 'substantially' bend DNA [Zweib, C. and Brown, R.S. (1990) Nucleic Acid Res. 18, 583-587]. These results also demonstrate that the TFIIIA/DNA complex has a large degree of conformational flexibility. Both DNA bending and conformational flexibility are structural features which may provide a key insight into the function of TFIIIA as a positive transcription factor.

Scattering Studies of the Isolated Red Blood Cell Skeleton, a Biological Two-Dimensional Polymer

AbstractWe studied the conformation of the membrane skeleton of human red blood cells (RBC) after detergent extraction of RBC ghosts, using video microscopy, light scattering, and synchrotronbased small angle X-ray scattering (SAXS). RBC membrane skeletons are two-dimensionally connected, triangulated networks of flexible, polyionic proteins. Immediately after extraction, the skeletons exhibited large-scale thermal undulations and deformed strongly in weak shear flow. Screening of electrostatic repulsion by immersion in high ionic strength buffer led to shrinkage, while the shell-like conformations and the flexibility of the skeletons were preserved. Under high ionic strength conditions (1 M monovalent salt), the static structure factor,S(q), showed two power law regimesS(q) ∝q−α, with α <≈ 2.0 in the range of wave vectors 4×10−4Å−1<g< 8×10−4Å−1, and α = 2.3 ± 0.1 in the range of wave vectors 8×10−4Å−1<q< l×10−1Å−1. The same power law behavior was observed in low ionic strength buffer (25 mM salt) forq< 2×10−3Å−1. This result is not consistent with the occurence of a crumpling transition during skeleton shrinkage. The observed form of the static structure factor, with a transition between two regimes with different power law exponents, presents evidence for the theoretically predicted flat phase of 2D-polymers.

A nuclear localization signal binding protein in the nucleolus.

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.

A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid

A modification of a protein-binding method (B. L. Brown et al., 1971, Biochem. J. 121, 561, 562) for measurement of adenosine 3′,5′-cyclic monophosphate (cAMP) in cell-culture supernatants and urine is described. With filtration over glass-fiber filters and a semiautomatic cell harvester instead of charcoal precipitation as in the original method, free [ 3H]cAMP tracer was rapidly and uniformly separated from that which was protein-bound. Sensitivity was increased with a high ionic strength buffer and a tritiated cAMP tracer with high specific activity. There was good agreement between cAMP values obtained with the protein-binding method and commercially available radioimmunoassays that were used as reference methods. cAMP could be determined accurately over the range 0.15 – 8.0 pmol/sample. More than 500 samples could be assayed in duplicate or triplicate in less than 6 h.

Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0 100 mM KC1. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KC1 concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent s 20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7 500 mM KC1) LTP sedimented with an apparent s 20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7 500 mM KCl which yielded a particle M r = 887000. Circular dichroism spectra of monomeric LTP sample revealed 6% α-helix, 49% β-sheet, 7% β-turn and 35% random coil while aggregated LTP contained 13% α-helix, 66% β-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.

Porphyrin intercalation and non-specific ‘edge on’ outside binding to natural DNA

A theoretical two-mode binding model for porphyrin binding to natural DNA is presented. One of the binding modes is assumed to be base sequence specific with binding sites n base-pairs long. The other binding mode has binding sites which consist of only one base-pair and can involve cooperativity. The model fits satisfactorily to data for H2TMPyP-4, Cu(II)TMPyP-3 and Cu(II)TMPyP-4 binding to calf thymus DNA in both a high (mu congruent to 1.0 M) and a low (mu congruent to 0.2 M) ionic strength buffer. The results show that the fraction of porphyrin bound in the non-specific mode reaches a maximum at certain input DNA to porphyrin concentrations ratios. The value of this maximum decreased, and its position shifted to higher DNA to porphyrin concentration ratios for binding in the high ionic strength buffer. The value of the cooperativity parameter obtained through the fitting process suggests that the non-specific binding is positively cooperative. The results are compared with the data analysed using other techniques.

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form.

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.

Human Urinary Trypsin Inhibitor (Urinastatin)-Like Substance in Mouse Kidney and its Relationships to Mouse Kidney Kallikrein

Mouse kidney contains urinastatin (UT)-like immunoreactive substances with trypsin inhibitory activity. Immunohistochemical studies show that these UT-like substances are localized in the same region as kidney kallikrein, i.e. in the distal tubules. Sephadex column chromatography of mouse kidney extract using 0.1M NaCl as the eluent yielded fractions (C.F.) containing both UT-like and kallikrein-like material. In these fractions (C.F.), the removal of UT-like material caused a concomitant decrease in kallikrein-like activity and vice versa. However, when the kidney extract was eluted with an acidic buffer of high ionic strength, the fractions containing both UT-like and kallikrein-like substances were not observed. These results suggest that these two components are intimately bound to one another. The kallikrein activity responded differently to pH, to metal ions (zinc and copper), and to the sodium/potassium ratio, depending on the concomitant presence or absence of UT-like material. These findings suggest that kallikrein activity in kidney tissue is modified by the presence of an UT-like substance.

Purification of a Dithiothreitol-Sensitive Tetrameric Protease from Spinach PS II Membranes

A protease with a tetrameric quaternary structure was extracted with 1 M NaCl from spinach PS II membranes and purified by hydrophobic, anion-exchange and gel-filtration chromatography using only buffers of high ionic strength. (...)

Heat-stable protein that stimulates acid alpha-glucosidase.

A hot-water extract of bovine spleen and guinea pig liver exhibited the ability to enhance acid alpha-glucosidase activity, with methylumbelliferyl alpha-glucoside, glycogen or maltose as substrate. The level of activator required for maximal stabilization was similar for all three substrates, indicating direct action on the enzyme rather than on substrate. The stimulator was partially purified by chromatography with gel-permeation (apparent Mr 20,000-24,000), ion-exchange and C4 reverse-phase columns. It was retained by a narrow-pore dialysis tubing and destroyed by treatment with Pronase, and is presumably a protein. The stimulating protein protected the enzyme against denaturation by heat or incubation with a buffer of high ionic strength in the absence of substrate. RNA inhibited the enzyme, and the activator protein was able to counteract the effect. Activating material was found in a variety of mouse and rat tissues, as well as human urine.

Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories

A procedure that is sufficiently simple and economical for use in clinical and public health laboratories for producing and purifying filamentous hemagglutinin (FHA) and determining antibodies to this major antigen of Bordetella pertussis in serum is described. High yields of FHA (40 to 80 mg/liter) were obtained in the supernatant by cultivating B. pertussis in modified CL medium. The FHA antigen was separated from pertussis toxin (PT) and other antigens by chromatography on hydroxylapatite. Removal of residual PT activity in the FHA fraction was effected by affinity absorption of PT with Fetuin immobilized to Sepharose 4B. The FHA was used as the antigen for determining titers of immunoglobulin G (IgG), IgA, and IgM to FHA in sera of patients with pertussis by an improved enzyme-linked immunosorbent assay. Development of the interfering background color commonly observed in conventional FHA enzyme-linked immunosorbent assay procedures was eliminated by washing the reaction wells with a buffer of high ionic strength before adding the peroxidase conjugates. In the absence of nonspecific background color, the reaction endpoints were easy to read. The FHA prepared by the procedure described was identical to a reference preparation of purified FHA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and serological specificity assays. High yields of FHA were obtained from all four strains of B. pertussis tested in this study, indicating that the procedure for enhanced production of FHA may be generally applicable to other strains of B. pertussis. Results from tests of 50 serum specimens with clinical information on pertussis for FHA and PT antibodies by the assay procedures described exemplified the usefulness and caveats of serodiagnosis for pertussis.

Formation of new quasi-crystalline ordered aggregates by gizzard myosin.

Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 mM KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl2 and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-S1 and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2 M KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg2+ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 mM MgCl2, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.

Conformational properties of streptokinase

The conformational properties of streptokinase (SK) have been assessed by the techniques of differential scanning calorimetry, circular dichroism (CD), and through a combinational approach employing several algorithms which are predictive of secondary structural characteristics. In low ionic strength buffers, SK undergoes a reversible two-state thermal transition with a temperature of maximum heat capacity (Tm) of 46.1 +/- 0.9, a delta Hcal of 98 +/- 11 kcal/mol and a delta Hcal/delta HvH of approximately 1. In high ionic strength buffers, similar calorimetric properties were obtained with the exception that the delta Hcal/delta HvH values were considerably less than 1, indicating the existence of an additional irreversible thermally induced alteration in the molecule, most likely resulting in its aggregation. The effect of pH on the thermal unfolding properties of SK was determined. The results demonstrated that single two-state thermal transitions were obtained, with progressively decreasing Tm values, as the pH was reduced from 6.4 to 3.4, indicating a destabilization of the entire molecule at reduced pH. In the alkaline region, between pH 8.4 and 9.4, stabilization of a separate region of the molecule was obtained, as evidenced by an increase in the delta Hcal/delta HvH to values approximating 2. CD analysis was performed in order to estimate secondary structural characteristics of SK. The best fit of secondary structural parameters to the experimental CD spectrum provided estimates of 17% helices, 28% beta-sheet, 21% beta-turns, and 34% disordered structures. Both the intensity of the spectral band at 208 nm and the level of antiparallel beta-sheet strongly suggest that SK is an alpha + beta protein.

Enrichment of Right-Side-Out Trypanosoma cruzi Plasma Membrane Vesicles

A simple method to prepare a high yield of Trypanosoma cruzi plasma membrane vesicles (PMV) from epimastigotes and metacyclic trypomastigotes is described. The method may be applicable to other protozoa. Solid-phase immunoassay to bind surface T. cruzi epitopes showed that this preparation was enriched with 80-82% PMV and that most of these were right-side out (81-92%). The method was based on the extraction of extrinsic proteins and subpellicular tubules with mild high and low ionic strength buffers without detergents (pH 7.4) and on the differential centrifugation of PMV based on their specific density (1.049 g/ml, 4 degrees C). Transmission electron microscopy of PMV pellets showed a heterogeneous population of vesicles without other significant cytoskeletal contaminants. T. cruzi PMV were also enriched with an ouabain- and oligomycin-insensitive magnesium-ATPase and contained an adenylyl cyclase, preserved for at least 3 months at -70 degrees C in storage buffer. Measurements of the [14C]-dextran and the 3H2O space indicated that T. cruzi PMV were not sealed, explaining why Lubrol PX and NaF failed to stimulate the adenylyl cyclase activity further and why T. cruzi PMV were unable to concentrate 86Rb in flow dialysis assays. No detectable DNA and RNA was found. The preparation was not capable of removing 51Cr or [3H]glucosamine from live L6 myoblast surfaces in physiologic conditions and acid phosphatase was extracted by this method. The contaminating fraction (18-20% by immunoassay) consisted of endoplasmic reticulum membranes with NADH oxidase activity and of kinetoplast membranes with cytochrome c oxidase and oligomycin sensitive magnesium-ATPase activity. The biologically active T. cruzi PMV retained the ability of living forms to trigger the alternate pathway of complement by releasing the Bb activation fragment from human Factor B.