Investigating the enzyme-substrate interactions of cytochrome c oxidase: a computer-assisted approach [abstract]

Although the enzyme cytochrome c oxidase (COX) is critical to respiration and has been studied extensively, the interactions between this enzyme and its substrate cytochrome c are still not very well understood. We employed a computer assisted approach to study these interactions. We used the Swiss-pdb v 2.5 computer programme, which measures the percentage accessibility of residues and the online server ANOLEA, which calculates the non-local energy of residues in a polypeptide chain, to analyze the respective molecular structures of cytochrome c and COX. The accessibility studies showed that, compared to the oxidized, the reduced form of cytochrome c normally had the greater proportion of more highly accessible residues: for reduced cytochrome c, 7 of the 8 residues exhibiting 55 percent accessibility were lysines. Interestingly enough, lysine 13, shown by other studies to be important for substrate binding, was not significantly accessible. For COX, neither asparate 158 nor glutamate 198 residues, reported to be important for substrate binding and catalysis, were significantly accessible either. The energy studies showed that whereas oxidized cytochrome c was a stable structure of low energy, approximatley 81 percent of the protein was reduced. For COX, a few small regions, including 4 residues in the vicinity of CuA, which functions as the enzyme's electron entry port, were of high energy. Since lysine 13, aspartate 158 and glutamate 198 are known to play important roles in enzyme-substrate interactions, it must be that these residues become more accessible when the two proteins interact. The results of the accessibility studies therefore appear to suggest that COX employs a mix of induced-fit and strain mechanisms when it binds substrate. On the basis of the energy studies, we conclude that the structure of reduced cytochrome c (the substrate) resembles a high energy transition-state intermediate and that when this protein binds and reduces oxidized COX, the structures of both proteins are stabilized. (AU)

In vivo studies of amino acid, water and electrolyte transport in the small intestine of normal and magnesium depleted rats

Net amino acid, water and electrolyte transport from the small intestine in vivo was studied in normal and magnesium depleted rats, by a single pass perfusion technique. The net absorption of the amino acids, alanine and lysine increased with increasing concentration of the amino acid in the perfusion solution, within the concentration range studied (10 and 50mM). In all cases alanine was absorbed faster than lysine. Dietary magnesium depletion, lasting for a period of twenty eight days did not affect the net rate of transport of these two amino acids in any of the regions of the small intestine which were studied. In general, the presence of amino acids increased the absorption of water by comparison with that from saline. However, lysine at a concentration of 50mM tended to inhibit water absorption by comparison with that observed in the presence of alanine at 50mM. Magnesium depletion did not in general affect net transport rates of water in the presence and absence of amino acids. However, when alanine at a concentration of 50mM was perfused, water absorption was inhibited in the magnesium depleted rats. The presence of amino acids did not affect the transport of sodium in any of the regions of the small intestine. However, magnesium depletion did severely inhibit sodium transport, especially in the presence of alanine at a concentration of 50mM in all three regions of the small intestine. Amino acids stimulated the absorption of chloride by all segments of the small intestine by comparison with that from saline alone. Magnesium depletion however, significantly reduced chloride ion absorption by all three segments. Good correlations were found between the transport rates of sodium and chloride, sodium and water, and total solute and water in all three regions of the small intestine (AU)

Dietary deficiency and pre-eclamptic toxaemia: a preliminary communication

Pre-eclamptic toxaemia is a major obstetric and neonatal problem in the Caribbean. It is associated with a high incidence of mortality and morbidity. The cause of pre-eclamptic toxaemia is not known. It is postulated that dietary deficiency in lysine and tryptophon may be the factors. A preliminary clinical trial is being conducted and a more complete survey is contemplated, depending on funds (AU)

Stimulation of insulin secretion in vitro by essential aminoacids

Leucine was the only essential aminoacid to stimulate insulin release from rabbit pancreas in vitri in the absence of extracellular glucose. In the presence of 1.5 mg glucose, per ml leucine, arginine, lysine and isoleucine were effective stimuli of insulin release (Summary)

The measurement of total lysine turnover in the rat by intravenous infusion of L-[U-14C] lysine

Rats were infused intravenously for periods up to 7 hr with L-[U-14C]lysine. After about 4 hr the specific activity of free lysine in the blood reached a plateau. From the value of the plateau specific activity it is possible to calculate the total lysine flux into or out of the blood stream. In normal rats the flux is equivalent to a total protein turnover of 25-30 g kg-1 day-1. In male, but not in female rats the total flux decreased significantly with increasing body weight or age. Six weeks of protein depletion caused a decrease of 30 percent in the flux. Alloxan diabetes in a small number of rats caused no significant change. After the first « hr of the infusion the specific activity of the mixed serum proteins increased almost linearly. From the rate of increase and the specific activity of the free lysine at plateau, an approximate estimate can be made of the renewal rate of the serum proteins. This estimate must be too low, because the specific activity of the free amino acid at the site of synthesis, e.g. in the liver cell, must always be lower than in serum. The validity of the continuous infusion method is discussed in relation to other methods of measuring total amino acid or protein turnover.(AU)

Lysine turnover in man measured by intravenous infusion of L-[U-14C] lysine

Lysine turnover was measured in three adult subjects by intravenous infusion of L-[U-14C]lysine for periods of 27-30 hours. A plateau specific activity of free lysine was reached in the plasma after c. 12 hours. The total turnover rates, calculated in terms of g protein.kg body weight-1 day-1 were 1.56 in an adult aged 50, 3.24 and 3.07 in two young men 19 years old. The results are discussed in relation to previous measurements of total amino acid turnover in man made by different methods. They fall within the range found by the methods which are theoretically the most satisfactory. (AU)

Observations on the mechanisms of adaptation to the low protein intakes

Experiments are described which attempt to throw light on the mechanism by which animals and man can adapt to low protein intakes. In the rat, studies by constant infusion of labelled amino acid have shown that in the protein depleted animal there is a small reduction in the total protein turnover: this, however, is not enough to account for the great reduction in urinary nitrogen output. Constant infusion and single injection experiments agree in showing that in rats on a low protein diet there is a change in the pattern of protein turnover: synthesis of carcass protein (muscle and skin) is reduced, while that of liver protein is well maintained. The preservation of synthesis in liver seems to depend partly on increased re-utilization of amino acids liberated by the catabolism of tissue protein. This economy may be brought about by adaptive enzyme changes -decreased activity of the urea cycle enzymes and increased activity of amino acid activating enzymes in the liver. These changes, previously described by others in the rat have been shown to occur in the human liver also. Studies in human infant with 75selenium-labelled methionine provide some support for the concept that when the protein intake is limited, turnover is preferentially maintained in the liver. However, not all liver-produced proteins behave in the same way; studies of albumin kinetics in infants show that when the protein intake is altered, there is a rapid change in the rate of albumine synthesis, together with a redistribution of albumin between intra and extravascular spaces. Later and more slowly occurs a change, presumably compensatory, in the rate of albumin catabolism. Hormonal changes may play a part in these adjustments. Increased cortisone and decreased insulin activity would have the effect of promoting amini acid uptake at the expense of muscle. It is concluded that the net nitrogen loss which occurs when the protein intake is reduced results simply from the time-lag before the adaptive mechanisms come into play, and therefore cannot logically be regarded as the loss of reserve protein. The practical implications of this concept are discussed (Summary)

The micro-measurement of lysine in the Cartesian diver

0.5-1.0ul. of solution containing lysine is mixed in silicone-coated divers (Waterlow & Borrow, 1949) with 2 ul. of a suspension of L-lysine decarbolylase (Sigma Chemical Co.). Evolution of CO2 is complete after 1 hr. By this method it is possible to estimate 0.01 uM lysine with an accuracy of about 5 percent. The method has been applied to the measurement of the turnover rate of free lysine in the rat during constant intravenous infusion with 14C-lysine. Serial blood samples are taken from the tail to give 100 ul. of plasma. After deproteinazation with picric acid the free amino acid extract is evaporated to dryness and taken up in 5 ul. of acetate buffer pH 5. This provides enough material for two duplicate measurements of lysine content and for measurement of radioactivity (AU)