Autocatalytically fragmented light chain of botulinum a neurotoxin is enzymatically active

The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc retained 100% of its original catalytic activity against a SNAP-25-derived synthetic peptide substrate. In the presence of zinc chloride, when >95% of the light chain had undergone autocatalytic fragmentation, the preparation retained 35% of its original catalytic activity. On the other hand, in the presence of glycerol, the light chain did not display autocatalysis and retained 100% of the original activity. These results suggest that the activity loss by incubation with zinc was not a direct consequence of autocatalysis and that the environment of the active site was not affected significantly by the fragmentation. The optimum pH 4.2-4.6 for autocatalysis was different than that (pH 7.3) for intrinsic catalytic activity. Inhibition of autocatalysis at low pH by a competitive inhibitor of catalytic activity rules out the presence of a contaminating protease but suggests a rate-limiting step of low pH-induced conformational change suitable for autocatalysis. Our results of LC concentration dependence of the fragmentation reaction indicate that the autocatalysis occurs by both intramolecular and intermolecular mechanisms.     

Enzymatic autocatalysis of botulinum A neurotoxin light chain

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

A spectrophotometric study of the denaturation of deoxyribonucleic acid in the presence of urea or formaldehyde and its relevance to the secondary structure of single-stranded polynucleotides

1. The thermal denaturation of DNA from rat liver was studied spectrophotometrically. In sodium phosphate buffers denaturation led to a single-stranded form having, at 25 degrees , about 25% of the hypochromism of the intact double helix. 2. The hypochromism of the denatured form was the same in 1mm- as in 10mm-sodium phosphate buffer and was scarcely affected by reaction with formaldehyde. The hypochromism was decreased by about 40% in the presence of 8m-urea. 3. The hypochromism of denatured DNA at low ionic strengths was about the same as that of fragments of reticulocyte ribosomal RNA that were too short to form double-helical secondary structure and about the same as that of RNA after reaction with formaldehyde. 4. The spectrum of DNA was slightly affected by the presence of 8m-urea or 4m-guanidinium chloride. The differences in the spectrum of the native and denatured forms of DNA in 0.1m-sodium phosphate buffer, in 8m-urea-10mm-sodium phosphate buffer and in 4m-guanidinium chloride-10mm-sodium phosphate buffer, pH7.6, were similar but not identical. 5. Denatured rat liver DNA appears to have no double-helical character at 25 degrees in 10mm-sodium phosphate buffer, pH7.6; increasing the buffer concentration to 0.1m leads to a more compact form in which about 40% of the residues form base pairs.     

Effect of hydroxypropyl methylcellulose and hydrogenated castor oil on naproxen release from sustained-release tablets

The effect of the concentration of hydrophilic (hydroxypropyl methylcellulose [HPMC]) and hydrophobic (hydrogenated castor oil [HCO]) products, fillers (lactose and dibasic calcium phosphate), and buffers (sodium bicarbonate, calcium carbonate, and sodium citrate) on naproxen release rate was studied. Matrix tablets were prepared by double compression, andIn vitro dissolution tests were performed. The dissolution results showed that an increased amount of HPMC or hydrogenated castor oil resulted in reduced drug release. The inclusion of buffers in the HPMC matrix tablets enhanced naproxen release. For HCO tablets, only sodium bicarbonate enhanced naproxen release. The presence of lactose on HPMC matrix tablets did not show a significantly different result from that obtained with the formulation containing dibasic calcium phosphate as a filler. However, for the tablets containing HCO, the presence of lactose significantly enhanced the naproxen release rate. The matrix-forming materials in this study were suitable for use in sustained-release tablets containing naproxen. The drug release can be modulated by adding suitable amounts of diluents and buffers.     

Superoxide,amine buffers and tetranitromethane: A novel free radical chain reaction

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.     

Botulinum Neurotoxin Types A,B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by <Emphasis Type="Italic">O</Emphasis>-Phenanthroline–Dithiothreitol,and Association of the Dinucleotides NAD<Superscript>+</Superscript>/NADH with the Heavy Chain of the Three Neurotoxins

The first evidence of autoproteolytic activity of the ~50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their ~100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183–1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249–Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221–231; Ahmed et al. (2003). Biochemistry 42:12539–12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (–P₁–P₁′ –) in one serotype (except Asp–Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249–Tyr250, Gln258–Ser259 and Ile243–Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT’s structure–function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418–Thr419 or Phe422–Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT’s entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs—the 9-residuelong motifs present on the NT’s natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD⁺/NADH associated with the NTs type A, B and E (2–3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase—a representative NAD⁺/NADH binding protein.     

Some factors that influence the nonenzymatic glycation of peptides and polypeptides by glyceraldehyde

The rate of reaction of glyceraldehyde with a series of peptides was found to be dependent on their amino acid composition, sequence, and chain length. The presence of a histidine near the NH₂-terminal increased the rate of glycation, whereas the presence of a carboxyl group near the reaction site led to a decrease in reaction rate. In general, tripeptides reacted faster than dipeptides, and dipeptides reacted faster than amino acids. Sodium phosphate and 2,3-diphosphoglycerate enhanced the rate of reaction of glyceraldehyde with all the dipeptides tested. Sodium chloride inhibited the reaction in phosphate buffer, but not in HEPES buffer. The NH₂-terminal heptapeptide from the -chain of human hemoglobin A (HbA), where histidine is the second residue, reacted with glyceraldehyde faster than the NH₂-terminal hexapeptide from the -chain. The glycation of tetrameric human Hb by glyceraldehyde was found to be dependent on the ligation state of the protein since deoxy-HbA reacted about 50% more with glyceraldehyde than did liganded HbA. The enhanced glycation of deoxy HbA was mainly attributable to the more extensive reaction at the NH₂-terminal of the -chain. The presence of a histidine adjacent to the NH₂-terminal at this site may facilitate the Amadori rearrangement. The glycation of horse Hb in which the second residue is glutamine was not increased under anaerobic conditions.     

Expression, purification, and characterization of Clostridium botulinum type B light chain

A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50 kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS-polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8 kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research.     

Studies on the sidedness of the human red blood cell membrane: preparation of stable azo-erythrocytes

Para-diazobenzenesulfonic acid (DBSA), a non-penetrating compound, which reacts with proteins of the external surface of intact cells, was used for the preparation of AZO-erythrocytes. When coupling of p-diazobenzenesulfonic acid to human red blood cells was carried out in phosphate-buffered saline (PBS), the cells lysed within one hour, before the completion of the reaction. Elimination of sodium chloride from the reaction mixture allowed the completion of coupling by prolonging the survival of red blood cells in he diazonium salt from one hour to three hours. The survival of human erythrocytes could be further prolonged to seven hours by carrying out coupling in the presence of cyclic 3',5'-guanosine monophosphate (cGMP). Azo-erythrocytes prepared in the absence of cyclic GMP and washed free of the diazonium salt lysed in phosphate-buffered saline, but remained stable for hours in isosmotic phosphate buffers devoid of sodium chloride ions. Under identical conditions, azo-erythrocytes prepared in the presence of cyclic GMP remained stable for two days and were suited for studies on the functional and structural aspects of red blood cell membrane.     

Myocardial muscarinic acetylcholine receptor: Choline and tris unmask heterogeneity of antagonist binding sites

The binding properties of myocardial muscarinic acetylcholine receptors are altered in the presence of choline or Tris. The binding of the antagonist [³H]quinuclidinyl benzilate is reduced in the presence of choline or Tris buffer, when compared to parallel determinations in a physiologic salt solution or phosphate buffer. Scatchard analysis indicates the reduced binding is due to a decrease in the apparent number of receptor sites. Experiments with other organic buffers exclude the possibility that the reduced binding in Tris is due to the absence of sodium ions. In the presence of choline or Tris up to 45% of the receptors are not accessible to [³H]quinuclidinyl benzilate. The remaining sites maintain their high affinity for the antagonist. A heterogeneity of antagonist sites is evident.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.     

Application of frozen thin sectioning immunogold staining to the study of the developing neuroepithelial basal lamina

Summary In order to examine the deposition of basal lamina components in the developing neurocpithelium, a technique for frozen thin sectioning and immunogold staining of early embryonic tissue was developed. Different fixatives and buffer systems were evaluated to determine which best retained immunoreactivity and satisfactory ultrastructure of day 9 and 10 mouse embryos. Fixation in sodium phosphate and sodium bicarbonate buffers did not retain antigenicity, and incubations in TBS (Trishydroxymethyl-aminomethane buffered saline) in an effort to restore immunoreactivity were similarly unsuccessful. Fixation in sodium cacodylate buffer, however, did retain the antigenicity of basal lamina components; the pattern of type IV collagen and laminin distribution was clearly determined. These results represent the first report of on-grid immunocytochemistry of carly embryonic material.     

Superoxide, amine buffers and tetranitromethane: a novel free radical chain reaction

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.     

The Effect of Phosphate Buffer on the Physiology of the Lichen Evernia prunastri

The physiological consequences of incubating either fresh ordesiccated thalli of Evernia prunastri in phosphate buffer orwater, in the presence or absence of added urea, was investigated.Phosphate buffer, with or without added urea, induced an immediateand sustained inhibition of photosynthesis. This was enhancedby prior desiccation. Urea in water also caused a reductionin photosynthesis but had little effect on respiration, whichwas initially enhanced by phosphate buffer but subsequentlydeclined. Release of intracellular K indicated a slower butsubstantial loss of membrane integrity in the presence of phosphatebuffer or, to a lesser extent, urea. Intracellular Na concentrationsrose initially on incubation in sodium phosphate buffer andthen declined, implying the occurrence of membrane damage. Urea-inducedurease activity was sustained in the presence of dithiothreitolwhen expressed on a unit protein basis. However, a decline wasobserved when results were calculated on a thallus dry weightbasis. The previously reported loss of urease activity on prolongedincubation in phosphate buffer is now suggested to be a consequenceof general buffer-induced damage rather than a specific urea-inducedsynthesis of inhibitory phenolic compounds. Evernia prunastri, cation location, lichen phenols, phosphate buffer, photosynthesis, respiration, urease activity     

Isolation and properties of an aminopeptidase from Bacillus licheniformis

An extracellular aminopeptidase, purified 465-fold from culture filtrates of Bacillus licheniformis, was found to be a metalloenzyme consisting of a single peptide chain. Sedimentation equilibrium yielded a molecular weight of 43,270 and two polyacrylamide electrophoretic procedures gave values of 37,500 and 36,000, respectively. The activity of the enzyme was inhibited severely by 1,10-phenanthroline and to a lesser extent by EDTA, cyanide, and fluoride. The addition of Co²⁺ ions greatly stimulated enzymatic activity, but analysis of the purified enzyme revealed the presence of zinc, not cobalt, in stoichiometric quantities. Moreover, the ratio of zinc to protein was found to increase during fractionation, reaching a final value corresponding to 1 g-atom/mol. The aminopeptidase possessed characteristics of a euglobulin, sparingly soluble in water and dilute buffer solutions, but soluble in buffers containing higher concentrations of salts. Both activity and pH optimum were substantially influenced by ionic strength; as the latter was increased over the range from 0.01 to 0.1, activity increased and the pH optimum was shifted to more acidic values. Enzymatic activity was affected by the identity of the buffer, being markedly greater in Tris-HCl than in sodium barbital and strongly inhibited by phosphate. The Bacillus aminopeptidase hydrolyzed substrates with unsubstituted amino groups of the l configuration, including dipeptides, aminoacylnaphthylamides, and amino acid amides.     

Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.     

Separation and characterization of heavy and light chains from Clostridium botulinum type C toxin and their reconstitution

Clostridium botulinum type C toxin consists of a heavy and a light chain with molecular weights of 98,000 and 53,000, respectively, which are linked by one disulfide bond. The two components were separated from each other by quaternary aminoethyl Sephadex A-50 column chromatography by stepwise elution with NaCl in 27.5 mM borax-45 mM sodium dihydrogen phosphate buffer, pH 8.0, containing 5% 2-mercaptoethanol at 0 degrees C. The purified components had different amino acid compositions and antigenicities, and the toxicity of the toxin was neutralized completely by either anti-heavy chain Fab or anti-light chain Fab. the two components could be reconstituted to form an active molecule with recovered toxicity which varied according to the method used. Maximum recovery was obtained in a system in which the intersubunit S--S bond was first formed in the presence of high concentration of neutral salts, after which the concentration of salt was gradually decreased. The reconstituted preparation was highly toxic and had the same properties as the parental toxin on chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunodiffusion. By the use of three perturbants, the fractions of exposed tryptophans and tyrosines of the preparation were found to be almost the same as that of the parental toxin.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> Heat Stable (STa) Enterotoxin and the Upper Small Intestine: Lack of Evidence in Vivo for Net Fluid Secretion

Heat stable (STa) enterotoxin from E. coli reduced fluid absorption in vivo in the perfused jejunum of the anaesthetized rat in Krebs-phosphate buffer containing lactate and glucose (nutrient buffer), in glucose saline and in glucose free saline. Bicarbonate ion enhanced fluid absorption of 98 ± 7 (6) μl/cm/h was very significantly (P < 0.0001) reduced by STa to 19 ± 4 (6) μl/cm/h, but net secretion was not found. When impermeant MES substituted for bicarbonate ion, net fluid absorption of 29 ± 3 (6) μl/cm/h was less (P < 0.01) than the values for phosphate buffer and bicarbonate buffer. With STa in MES buffer, fluid absorption of 3 ± 2 (6) μl/cm/h was less than (P < 0.001) that in the absence of STa and not significantly different from zero net fluid absorption. E. coli STa did not cause net fluid secretion in vivo under any of the above circumstances. Neither bumetanide nor NPPB when co-perfused with STa restored the rate of fluid absorption. In experiments with zero sodium ion-containing perfusates, STa further reduced fluid absorption modestly by 20 μl/cm/h. Perfusion of ethyl-isopropyl-amiloride (EIPA) with STa in zero sodium ion buffers prevented the small increment in fluid entry into the lumen caused by STa, indicating that the STa effect was attributable to residual sodium ion and fluid uptake that zero sodium-ion perfusates did not eradicate. These experiments, using a technique that directly measures mass transport of fluid into and out of the in vivo proximal jejunum, do not support the concept that E. coli STa acts by stimulating a secretory response.