Search

Using a chemical quench device, the rate of synthesis of carbamyl aspartate from the substrates aspartate and carbamyl phosphate was followed as a function of the time between mixing the enzyme with substrates and quenching with trichloroacetic acid. This function, which is linear at long times, shows (at 4 °C) a transient lag phase of product of roughly 10 ms. However, when the catalytic subunit (in which the enzymatic activity is desensitized) is used instead of the enzyme, the lag disappears. Therefore the lag seems to be associated with the control functions of the enzyme, i.e. to represent the allosteric transition involved in substrate-substrate (homotropic) co-operativity. Thus the relaxation time for the activation process is roughly 10 ms. The implications of these results are examined.

Dehydroacetic acid (DHA) labeled with C14 in four carbon atoms has been synthesized. DHA administered orally to rabbits at a dose level of 250 mg/kg did not increase the urinary excretion of glucuronides or ethereal sulfates. Small oral doses (20–70 mg/kg) of C14-DHA were not rapidly excreted. In 3–7 days rabbits excreted 70–80% of the dose in the urine, 7–10% in respiratory CO2, and 2–3% in the feces; 8–17% remained in the tissues. In 4–5 days, rats excreted 20–40% in the urine, 10–25% in respiratory CO2, and 10–20% in the feces; 5–26% remained in the tissues. In both rats and rabbits the highest radioactivity was found in the blood. Electrophoresis of human serum incubated with C14-DHA showed that DHA is mainly associated with serum albumin and to a lesser extent with serum globulins. Three metabolites of DHA have been isolated from rabbit urine: triacetic acid lactone (TAL); a hydroxy-DHA, probably 3-glycoloyl-6-methyl-2,3-dihydropyran-2,4-dione; and metabolite X, which is probably the salt of triacetic acid lactone 3-carboxylic acid. A further metabolite, compound Y, which is not a pyrone but probably a degradation product of DHA, was detected. DHA and hydroxy-DHA react with ammonium salts to form the corresponding 1′-imino derivative, imino-DHA and imino-hydroxy-DHA. After administration of DHA to rats and rabbits, the DHA and hydroxy-DHA occur in the urine partly free and partly as the imino derivatives. The urines of rabbits receiving small oral doses of C14-DHA contain an average of DHA (total) 5% of the dose, hydroxy-DHA (total) 20%, TAL 10%, urea 0.3%; metabolites X and Y were estimated to account for 20 and 15%, respectively. Similarly, rat urines contain DHA (total) 5%, hydroxy-DHA (total) 8%, TAL 1%, and urea 0.3%. At higher dose levels the percentages of dose excreted as DHA and hydroxy-DHA are markedly increased, and the percentages as metabolites X and Y markedly decreased. 2,6-Dimethyl-4-pyrone and its 3-carboxylic acid, deoxydehydroacetic acid; orcinol; malonic, succinic, oxalic, and acetic acids; and acetone were shown not to be urinary metabolites of DHA. DHA and hydroxy-DHA, but not TAL, were found in the feces and gut contents of rats after subcutaneous injection of DHA, suggesting that these two compounds are excreted in the bile. The pattern of excretion of metabolites after injection of DHA did not differ significantly from that following oral administration. C14-DHA was metabolized by rat liver, but not kidney, to hydroxy-DHA, TAL, and CO2. A pathway for the metabolism of DHA via hydroxy-DHA, compound X, TAL, and triacetic acid to acetoacetic acid and CO2 is suggested. Because of the reactivity of the keto group of the acetyl side chain and the tendency of DHA and hydroxy-DHA to combine with the amino groups of proteins and other essential compounds, the administration of DHA might have serious deleterious effects. The ultraviolet absorption spectra, color reactions, and chromatographic behavior of the metabolites of DHA and a number of other pyrones are given.

A simple method for the determination of tryptophan residues in proteins is described. The method involves (a) treatment of the protein with an excess of 2-hydroxy-5-nitrobenzyl bromide, (b) purification of the labeled protein by gel filtration and ethanol washing, (c) measurement of the absorbance at 410 mµ to determine the number of colored groups incorporated, and (d) a determination of the total protein content by conventional methods. The procedure has been shown to be accurate with a wide variety of proteins.

The effects on the function and conformation of α-lactalbumin after reaction with 2-hydroxy-5-nitrobenzyl bromide and its water soluble derivative dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide have been studied. Three of the four tryptophan residues of α-lactalbumin (Trp 26, Trp 104 and Trp 118) were accessible to the tryptophan reagents and the reaction mixtures were complex. The labelling process could not be made more selective by changing the experimental conditions and to determine the importance of each of the accessible tryptophan residues an attempt was made to separate α-lactalbumin labelled uniquely at Trp 26, Trp 104 or Trp 118. A partial separation was possible by ion-exchange chromatography and three preparations thus isolated were subjected to kinetic and conformational studies. The results obtained suggest that when Trp 26, Trp 104 or Trp 118 is alkylated with the 2-hydroxy-5-nitrobenzyl group, α-lactalbumin loses its specifier protein activity. The labelling of tryptophan did not cause a gross conformational change but a minor change may have occurred.

The reactivities of the tryptophan residues of bovine milk α-lactalbumin and hen egg-white lysozyme towards a specific tryptophan reagent, 2-hydroxy-5-nitrobenzyl bromide, have been studied. The side chains of the six tryptophan residues of lysozyme react in general agreement with its known three-dimensional structure. Three of the four tryptophan residues of α-lactalbumin (Trp 63 † , Trp 108 and Trp 123) react in a way that can be interpreted with reference to the proposed three-dimensional structure of this protein. The fourth residue (Trp 28), predicted from the proposed structure to be completely buried, is unexpectedly reactive to the reagent.

1. 1.|The reaction of α-lactalbumin with 2-hydroxy-5-nitrobenzyl bromide and its water-soluble derivative, dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide have been examined. 2. 2.|At pH 2.7 α-lactalbumin was exceptionally reactive. At low molar ratios of reagent to protein, three of the four tryptophan residues reacted, namely Trp 26, Trp 104 and Trp 118. Under the conditions used, all of the incorporated 2-hydroxy-5-nitrobenzyl (HNB) group could be accounted for as the two possible monosubstituted products of tryptophan. The amount of disubstituted tryptophan was very low and there was no evidence for the modification of amino acids other than tryptophan or for inter- or intra-molecular cross linkages. At very high molar ratios of reagent to protein, all of the tryptophan residues were disubstituted. 3. 3.|At pH 6.0, both of the tryptophan reagents were less specific than at pH 2.7 and a histidine residue reacted (His 32) in addition to Trp 26, Trp 104 and Trp 118. Further, of the HNB group incorporated, about 30% was labile at pH 8.5. 4. 4.|A general method for the isolation in high yields of tryptophan-containing peptides labelled with the 2-hydroxy-5-nitrobenzyl group is also described.

Received 17 October 1977 1. Introduction ATP affinity analogs are of special usefulness in the study of the binding and hydrolysis of ATP by myosin ATPase during the contraction cycle. A number of these ATP analogs have been synthe- sized and their reaction with myosin described [1-3]. It was reported [4] that the synthesis of a brominated adenosine-5' ester of maleic acid and pointed out its potential use as an active-site-directed inactivator of certain adenosine nucleotide dependent enzymes, attacking the phosphate binding site. In this report we describe an improved synthesis of a similar derivative, 2'-3' isopropylidene adenosine 5' dibromohydrogen succinate, and its reaction with rabbit skeletal myosin and its chymotryptic subfrag- ment-1. 2. Experimental

For the first time, the structural change associated with an allosteric transition has been monitored by X-ray solution scattering. The kinetics of the quaternary structure change of aspartate transcarbamylase were first slowed by using acetyl phosphate instead of carbamyl phosphate, and by the presence of 10% or 30% ethylene glycol. At 6.5 degrees C, the quaternary structure change was found to have a time constant of about 11 seconds. This appears to be larger than that obtained for the switching of homotropic co-operativity, measured by chemical quench under the same conditions.

We have studied the effects on the function and conformation of bovine α-lactalbumin after reaction with the protein-dissociating reagent maleic anhydride. The 13 amino groups of α-lactalbumin were accessible to the reagent and when all of these were maleylated a highly acidic and more expanded protein resulted. Despite these differences in physical properties, maleyl-α-lactalbumin was as effective as native α-lactalbumin in the lactose synthetase reaction. On the other hand, when α-lactalbumin was treated with trinitrobenzenesulphonic acid, inactivation occurred.

Triacetic acid lactone (TAL) labeled with C 14 in three carbon atoms has been synthesized. The C 14 of small oral doses of C 14 -TAL fed to rats (250–350 mg/kg) and rabbits (50–60 mg/kg) was mostly excreted in the expired air and urine within 24 hours. In 3 days, rats excreted 45–50% in the respiratory CO 2 , 20% in the urine, and 7% in the feces; 10–20% remained in the tissues. Rabbits, in about the same period, excreted 50–60% as CO 2 , 30% in the urine, and 4% in the feces; 10–20% remained in the tissues. With both rats and rabbits most of the radioactivity in the urine occurs as unchanged TAL (13–25% of dose) together with small amount of urea (2–11% of dose). In the urines of rabbits only, a third unidentified metabolite accounting for 10% of the dose, and probably a degradation product of TAL, was detected. Paper chromatography did not reveal the presence of DHA, hydroxy-DHA, or compound X in the urines of rats or rabbits dosed with C 14 -TAL. C 14 -TAL is more rapidly oxidized to CO 2 by rat kidney slices than by liver slices. DHA, hydroxy-DHA, and compound X were not detected as in vitro metabolites of TAL. Unlike DHA, TAL does not appear to combine with tissue proteins.