Formation of cyanate and carbamyl phosphate by electric discharges of model primitive gas

A mixed gas of nitrogen, carbon dioxide, and hydrogen was discharged over 100 ml of 0.2M NaHCO₃ solution in a 5 liter discharge apparatus which simulates the primitive Earth. The formation of cyanate, which is one of the possible primitive condensing agents, was demonstrated by the detection of [Cu(Py)₂] (NCO)₂ that was formed by the addition of copper sulfate-pyridine reagent to the solution. In a series of experiments the partial pressures of nitrogen and carbon dioxide in the starting gas were fixed at 10 cm Hg and 20 cm Hg, respectively, whereas that of hydrogen was varied between 5, 10, 15, 20, 25, and 30 cm Hg. The discharges were continued for one week. The rate of appearance of cyanate was strongly dependent upon the partial pressure of hydrogen. The maximum rate of the production of cyanate at the initial stage of the discharge was in the case of 10 cm Hg of hydrogen, in which condition the starting gas is in a predominantly oxidized state. In this case the concentration of cyanate reached about 0.012M after one day. Another discharge experiment was carried out with 0.2M phosphate solution, and the production of carbamyl phosphate was demonstrated through the formation of ATP by the incubation of the discharged solution with ADP and carbamyl phosphokinase.     

Constant AMP synthesis in aqueous solution by electric discharges

An electric discharge was made through a gas mixture of N₂ (7 cm Hg), H₂ (14 cm Hg) and CO₂ (14 cm Hg) over an aqueous solution (100 ml, pH 7.6) of adenosine (0.02 M) and phosphate (0.2 M) in a 5 liter vessel simulating primitive earth conditions. AMP was produced at a constant rate in the solution, and the yield reached about 2% in two months.     

ATP regeneration using immobilized carbamyl phosphokinase

A simple and efficient system for continuous ATP regeneration is described. The procedure is based on the enzyme-catalyzed reaction between carbamyl phosphate and ADP. The carbamyl phosphate was generated in situ by reaction between potassium cyanate and potassium phosphate. The enzyme, carbamyl phosphokinase, was isolated from extracts of Streptococcus faccalis and partially purified. Immobilization of the enzyme was achieved using glutaraldehyde-treated alkylamine glass giving 200–250 units of activity per gram of glass. A column of carbamyl phosphokinase on glass was used to form ATP continuously from ADP, phosphate, and cyanate and lost approximately 16% of the initial activity after 14 days operation at room temperature.     

Amino acids derivatives synthesis from nitrogen, carbon and water by electric discharges

Electric discharges between a pair of carbon electrodes were continued for 50 days in a vessel of 5 liters in volume which initially contained nitrogen at a pressure of 15 cm Hg and 200 ml of water. The pressure in the vessel was gradually increased to 60 cm Hg at the end of the run. Gas chromatographic analysis showed that the increase of the pressure mainly results from the production of hydrogen and carbon monoxide. The concentration of ammonia in the aqueous sample was increased to 0.05 M in 50 days of the discharge. After hydrolysis, glycine and serine were detected at the concentrations of 3.4 x 10(-3) M and 0.057 x 10(-3) M in the final solution, respectively, though glycine was found only at the concentration of 6 x 10(-6) M before hydrolysis. TLC analysis indicated the presence of hydantoic acid, N-formylglycine, diketopiperazine, and polymers of glycine.     

Carbonic anhydrase III inhibition in normocapnic and hypercapnic contracting mouse soleus

The physiological role of carbonic anhydrase III in slow-twitch skeletal muscle was investigated using isolated mouse soleus (N = 30) contracting once every 1.7 min for 75 min in Krebs-Henseleit solution gassed with either 95% oxygen - 5% carbon dioxide (normocapnia) or 90% oxygen - 10% carbon dioxide (hypercapnia). Each contraction was 500 ms in duration at 50 Hz. When muscles contracted in normocapnic solution (pH 7.42), the developed tension decreased an average of 6.1 +/- 0.8% over 25 min. For the next 50 min, 15 muscles remained normocapnic, while the remainder contracted in hypercapnic solution (pH 7.20). Tension decreased significantly more with hypercapnia. For the last 25 min, both normocapnic and hypercapnic muscles were divided into three treatment groups (N = 5). One group continued in the same environment, while acetazolamide (final concentration of 10(-5) M) was added to the bath of the second and sodium cyanate (final concentration of 10(-5) M) was added to the bath of the third group. Acetazolamide had no effect on tension in either carbon dioxide environment. Sodium cyanate significantly decreased tension from the hypercapnic control but had no effect in normocapnia. Thus carbonic anhydrase III inhibition with sodium cyanate increased the effect of hypercapnia implying that carbonic anhydrase III assists in the regulation of free hydrogen ion concentration in slow-twitch skeletal muscle.     

Hydrolysis of cyanate catalyzed by guinea pig and rat tissue extracts

The hydrolysis of cyanate to give ammonia and CO₂ catalyzed by extracts of liver and kidney from rats or guinea pigs is not due to the presence of cyanase as previously reported. Instead, the hydrolysis apparently results from the chemical reaction of cyanate with phosphate at pH 6.0 to give carbamyl phosphate which is subject to hydrolysis catalyzed by a phosphatase in the extracts.     

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.     

Gas Exchange of Algae: II. Effects of Oxygen, Helium, and Argon on the Photosynthesis of Chlorella pyrenoidosa

Changes in the oxygen partial pressure of air over the range of 8 to 258 mm of Hg did not adversely affect the photosynthetic capacity of Chlorella pyrenoidosa. Gas exchange and growth measurements remained constant for 3-week periods and were similar to air controls (oxygen pressure of 160 mm of Hg). Oxygen partial pressures of 532 and 745 mm of Hg had an adverse effect on algal metabolism. Carbon dioxide consumption was 24% lower in the gas mixture containing oxygen at a pressure 532 mm of Hg than in the air control, and the growth rate was slightly reduced. Oxygen at a partial pressure of 745 mm of Hg decreased the photosynthetic rate 39% and the growth rate 37% over the corresponding rates in air. The lowered metabolic rates remained constant during 14 days of measurements, and the effect was reversible after this time. Substitution of helium or argon for the nitrogen in air had no effect on oxygen production, carbon dioxide consumption, or growth rate for 3-week periods. All measurements were made at a total pressure of 760 mm of Hg, and all gas mixtures were enriched with 2% carbon dioxide. Thus, the physiological functioning and reliability of a photosynthetic gas exchanger should not be adversely affected by: (i) oxygen partial pressures ranging from 8 to 258 mm of Hg; (ii) the use of pure oxygen at reduced total pressure (155 to 258 mm of Hg) unless pressure per se affects photosynthesis, or (iii) the inclusion of helium or argon in the gas environment (up to a partial pressure of 595 mm of Hg).     

Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase

Brzozowski, Thomas H. (Stanford University School of Medicine, Palo Alto, Calif.), and Sumner M. Kalman. Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase. J. Bacteriol. 91:2286-2290. 1966.-Earlier studies have shown that the carbamyl phosphate synthesis from ammonia in cell extracts of wild-type Escherichia coli is due to at least two enzymes, acetokinase and the glutamine-dependent carbamyl phosphate synthetase. Partial purification of the glutamine-dependent carbamyl phosphate synthetase and acetokinase fails to separate from these enzymes this ammonia-dependent activity. An antibody to the partially purified acetokinase was prepared and used to determine the distribution of the ammonia-dependent activity in wild-type organisms and single-step arginine-uracil-requiring mutants with respect to the two enzymes. Such a study was possible because the antibody inhibits acetokinase but not the glutamine-utilizing carbamyl phosphate synthetase. Enzyme inhibition obtained by the stepwise addition of the antibody to cell extracts indicates that all of the ammonia-dependent carbamyl phosphate synthesis observed in the arginine-uracil-requiring mutants is due to a protein in the acetokinase fraction, presumably acetokinase itself, since acetyl phosphate and carbamyl phosphate synthesis were inhibited in a parallel fashion. In wild-type organisms, there is only partial inhibition of the ammonia-dependent activity, even when enough antibody is added to produce maximal inhibition of acetokinase. It is suggested that this residue is due to the glutamine-dependent carbamyl phosphate synthetase, for the ratio of the antibody insensitive to antibody sensitive ammonia-dependent activity present in cell extracts of the two wild-type organisms reported is qualitatively proportional to the level of carbamyl phosphate synthetase present relative to acetokinase.     

Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.     

Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit.

The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.     

Quantifying the allosteric properties of Escherichia coli carbamyl phosphate synthetase: determination of thermodynamic linked-function parameters in an ordered kinetic mechanism.

The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3(-)-dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent Km for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, Kia, in the ATP synthesis reaction, with UMP increasing Kia 15-fold and ornithine decreasing Kia by 18-fold. By contrast, only UMP substantially affects the Kia for MgATP in the ATPase reaction, increasing it by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Carbamyl Phosphate Inhibition of Nitrogenase of Clostridium pasteurianum

Carbamyl phosphate caused a maximal inhibition of 50% of the in vitro nitrogenase activity measured by acetylene reduction and dinitrogen reduction. The addition of 1 mM carbamyl phosphate to a N(2)-fixing culture caused a rapid decrease of 30% of the acetylene reduction activity and also repression of nitrogenase biosynthesis. However, carbamyl phosphate had no effect on the reductant-dependent adenosine triphosphate hydrolysis and H(2) evolution reactions catalyzed by nitrogenase. Studies on the binding of carbamyl phosphate to nitrogenase and each of its two components (azoferredoxin and molybdoferredoxin) indicated that optimal binding was obtained only in the presence of an operating nitrogenase system. Moreover, the binding seemed to be on the molybdoferredoxin component rather than azoferredoxin. From a Scatchard plot and a reciprocal plot of the data, the values of n = 2 and dissociation constant (K) of approximately 5 x 10(-5) M were obtained. The value for the dissociation constant was of the same order of magnitude as the endogenous level of carbamyl phosphate in a N(2)-fixing cell. The carbamyl phosphate pool in NH(3)-grown cells was twice that of N(2)-fixing cells.     

Study of the Formation Time of a Self-Sustained Subnanosecond Discharge at High and Ultrahigh Gas Pressures

The formation times of self-sustained subnanosecond discharges in nitrogen at pressures of 1?40 atm and in hydrogen at pressures of 1–60 atm are analyzed in terms of the avalanche model. In experiments, a subnanosecond voltage pulse with an amplitude of 102 ± 2 kV was applied to a 0.5-mm-long discharge gap with a uniformly distributed electric field (the curvature radii of both the cathode and anode ends were 1 cm). The rise time of the voltage pulse from 0.1 to 0.9 of its amplitude value was about 250 ps. Breakdown occurred at the leading edge of the pulse. The discharge formation time was measured at different gas pressures with a step of 5–10 atm. Analysis of the experimental results shows that, in nitrogen at pressures of 10–40 atm and in hydrogen at pressures of 20–50 atm, breakdown occurs earlier than the electron avalanche reaches its critical length and that the critical avalanche length lies in the range of (2–8) × 10^–2 mm, which is one order of magnitude shorter than the discharge gap length. This means that the avalanche–streamer model is inapplicable in this case. The fast formation of a conducting channel under these conditions can be explained by ionization of gas by runaway electrons. In this case, the conducting column develops as a result of simultaneous development of a large number of electron avalanches in the gas volume. An increase in the hydrogen pressure from 50 to 60 atm leads to an abrupt increase in the discharge formation time by about 50%. As a result, the growth time of the electron avalanche to its critical length becomes shorter than the discharge formation time. In this case, the electrons cease to pass into the runaway regime and the discharge is initiated from the cathode due to field emission from microinhomogeneities on its surface. Under these conditions, the discharge formation time is well described by the avalanche–streamer model.     

A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.     

Amino acids derivatives synthesis from nitrogen,carbon and water by electric discharges

Electric discharges between a pair of carbon electrodes were continued for 50 days in a vessel of 5 liters in volume which initially contained nitrogen at a pressure of 15 cm Hg and 200 ml of water. The pressure in the vessel was gradually increased to 60 cm Hg at the end of the run. Gas chromatographic analysis showed that the increase of the pressure mainly results from the production of hydrogen and carbon monoxide. The concentration of ammonia in the aqueous sample was increased to 0.05M in 50 days of the discharge. After hydrolysis, glycine and serine were detected at the concentrations of 3.4×10^–3 M and 0.057×10^–3 M in the final solution, respectively, though glycine was found only at the concentration of 6×10^–6 M before hydrolysis. TLC analysis indicated the presence of hydantoic acid, N-formylglycine, diketopiperazine, and polymers of glycine.     

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (K_i) of 2.6 × 10^?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [³²P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.     

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.     

Mass spectrometry as en ecological tool for in situ measurement of dissolved gases in sediment systems

Abstract The use of membrane-inlet quadrupole mass spectrometry, as a method for quantitative monitoring of dissolved gases in natural or semi-natural environments, is described. Its advantages over other methods lie in the fact that it provides an accurate, sensitive means for non-invasive, continuous analysis of several dissolved gases simultaneously. The potential of mass spectrometry as an ecological tool is illustrated by representative results from measurements made on undisturbed and experimentally amended estuarine and fresh-water sediments.
Dissolved gas profiles from the surface to a depth of 10 cm in the estuarine sediment showed that the dissolved oxygen decreased gradually until at 10 cm it was undetectable (< 0.25 μM); dinitrogen reached a maximum at 6 cm, where oxygen was 20 μM. In a fresh-water sediment, methane reached 1.5 mM at 10 cm depth. NO_x was also detected; quantitation of carbon dioxide necessitates a correction for the contribution of NO_x. Manipulation of conditions (gas phase, nitrogen and carbon sources) permitted ecological modelling.     

The reversible inhibition of exsheathment in some parasitic nematodes

• 1.1. Exsheathment of infective juveniles of Haemonchus contortus in bicarbonate-carbon dioxide buffer (gas phase, 40 per cent carbon dioxide in nitrogen) was inhibited by exposing the juveniles to 5 × 10⁻⁴ N iodine for 10 min at 25°C. This inhibition was decreased if the iodine-treated juveniles were exposed to hydrogen sulphide-water before they were stimulated to exsheath. The effect of iodine was also decreased when 0·01 M sodium dithionite was included in the stimulating medium.
• 2.Solutions of iodine and hydrogen sulphide had similar effects on infective juveniles of Trichostrongylus colubriformis which were stimulated to esheath in 0·01 N hydrochloric acid under a gas phase of 40 per cent carbon dioxide in nitrogen. With this species, however, the effect of hydrogen sulphide was often greater, and the inhibition caused by iodine could be completely reversed.
• 3.It is argued that these results support the hypothesis that, in order for infection to occur with juveniles which enter the host per os, the host must supply a stimulus which initiates the development of parasitic stages. Carbon dioxide seems to be the chief component of the stimulus provided by the host. It is postulated that the carbon dioxide reacts with receptors in infective juveniles. The results given in this paper support the hypothesis that suitably placed sulphydryl groups may be involved in the interacion of carbon dioxide with the receptor.