Essential and nonessential thiols of yeast hexokinase. Reactions with iodoacetate and iodoacetamide.

The reaction of yeast hexokinase with iodoacetate or iodoacetamide has been investigated in detail, using pure hexodinase B. Of the four thiols in each subunit of the molecule, two (the "apparently essential thiols") are alkylated rapidly at 35 degrees, and the enzymic activity is lost in parallel with their reaction. The other two thiols react subsequently to completion, but at a very much slower rate. In the conditions use, no other uptake of the reagent occurs elsewhere during these thiol alkylations. Electrophoretically homogeneous kialkylated and tetraalkylated protein species are formed, in the two stages of the reaction. The inactivating reaction at 35 degrees with the apparently essential thiols is second order. The rate constant increases with increasing pH, in the range pH 7.0-8.5, in a manner consistent with control of the reaction by a group with pKa of approximately 10. The absolute (pH independent) rate constant is of the same order as that for a normal thiol in model compounds. The availability of the apparently essential thiols appears to be associated with some conformational change in the molecule in the monomer form: it declines at high ionic strengths, is maximal at intermediate values where the dimer first dissociates, but is lowered in the dimer at very low ionic strengths. The reaction also shows a sharp temperature dependence: the dimer at 30 degrees (in constrast to 35 degrees) shows no availability of the apparently essential thiols. A similar transition to a state permitting fast inactivation is found with pH, above pH 8.5. The reaction of the two apparently essential thiols is strongly inhibited by glucose. ATP and ADP, and their Mg complexes, protect significantly, but less effectively than does glucose. The affinities of these substrates at the active site of the enzyme are measured in this protection system. These various reactions appear to be of value for identifying the cysteine-containing regions that are involved in the active center or in its maintenance in the structure.     

Motility pattern and lung respiration of embryonic chicks under the influence of L-thyroxine and thiourea

Pressure changes in the air cell and at the egg shell have been used to monitor respiratory and somatic movements of embryonic chicks. During the prehatching period a phase of reduced activity is observed. Pulmonary respiration is initiated during this phase. Exogenous L-thyroxine exerts an accelerating effect on the hatching process and on the onset of the phase of reduced motility and of lung respiration. In thiourea-treated embryos the opposite effects on the hatching process and on the motility and respiration pattern are registered. When, however, the egg shell above the air cell was sealed with glue, times of hatching and of the beginning of lung respiration were similar to those of controls, although pipping the egg shell occurred earlier than normal. It is suggested that the effects of L-thyroxine and thiourea on the hatching process are caused by a premature or delayed onset, respectively, of pulmonary respiration.     

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a₃ components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a₃ was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.     

Reaction of p-azidophenacyl iodoacetate, a photolabile reagent, with yeast alcohol dehydrogenase.

When yeast alcohol dehydrogenase (YADH) was incubated with one or two molar equivalents of the photolabile reagent p-azidophenacyl iodoacetate (1), 10–15% of the enzymatic activity was lost per mole of inhibitor incorporated, a result which suggests 1 may be modifying in a cooperative process both the Cys-43 and the Cys-153 groups found at each active site of the enzyme. YADH incorporated a maximum of 5.6 mol of 1 per mole of enzyme. When YADH was first carboxymethylated and then allowed to react with an excess of 1, 3.2–3.6 mol of 1 were incorporated into the enzyme with a corresponding loss of 4.0 mol of free sulfhydryl groups in the enzyme. Carboxymethylated YADH was reacted with one molar equivalent of ¹⁴C-1 and then was treated sequentially with hydroxylamine and pepsin. Cellulose phosphate chromatography of this peptic digest gave one major radioactive peak eluting in the region where peptic peptides of YADH known to be modified at the Cys-153 are found to elute. When carboxymethylated YADH was treated with one molar equivalent of 1 and then photolyzed, at least 18% of the 1 residues became covalently bound to a second site in the enzyme. This finding establishes that 1 is a useful reagent for investigating the three-dimensional structure of the active site of YADH. Furthermore, 1 should be suitable for investigations into a variety of biological systems.     

Bacteriophage formation without bacterial growth; the effect of iodoacetate,fluoride, gramicidin,and azide on the formation of bacteriophage

1. Iodoacetate, fluoride, and azide have been found to prevent the formation of phage and to inhibit the synthesis of ATP by Staphylococcus muscae. It is suggested that energy-rich phosphate is needed for the synthesis of phage. 2. Gramicidin prevented the formation of phage. 3. No differences were found between normal bacteria and phage-infected bacteria in the inorganic phosphate, adenosinetriphosphate, ribonucleic acid, and desoxyribonucleic acid content of the cells. 4. The mechanism of phage formation is discussed.     

Non-destructive measurement of dormant bud respiration rates

The lack of an indicator of the state of bud development during the dormant period has been a major difficulty in studying the effects of winter chilling on subsequent shoot growth and flowering. We considered that respiration rate (R _D ) might provide such an index, so developed a technique for the non-destructive measurement of the R _D of individual dormant buds of kiwifruit (Actinidia deliciosa). A closed configuration gas exchange system was used. The low R _D of dormant buds required the use of an unusually small system volume. As a consequence, it was necessary to modify the conventional closed system so that most of the system volume could be sampled for analysis. Increases in CO₂ concentration during a measurement were determined by injecting gas samples into a stream of air flowing through an infrared gas analyser. The technique was found to be reliable even at R _D as low as 20 pmol s-1. Error analysis showed that under normal operating conditions the coefficient of variation was approximately 3 %. With two operators, measurements could be made at the rate of one bud every four minutes. The ability to make non-destructive measurements has the advantage of enabling us to make sequential measurements on individual buds and monitor subsequent development. The system could be readily adapted to other woody fruit species, providing that gas-tight seals can be established on individual buds. Use of the system is illustrated by measuring the temperature response of the R _D of dormant buds grown under contrasting conditions. All buds showed increasing R _D with increasing temperature in the range 15 to 25 °C. Buds which had grown in the coolest region, where bud break is usually the earliest, had the highest R _D when measured during early spring. This revised version was published online in September 2006 with corrections to the Cover Date.