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Biomedical subjects

B Middleton

Publications and source records attributed to B Middleton.

119 records · Page 7Linked to original sources
PubMed1973

The oxoacyl-coenzyme A thiolases of animal tissues.

1. The activities and relative 3-oxoacyl-CoA substrate specificities of oxoacyl-CoA thiolase were determined in a large number of animal tissues. The relative activities with different 3-oxoacyl-CoA substrates varied widely in different tissues and, in addition, the activity as measured with acetoacetyl-CoA (but not with other longer-carbon-chain acyl-CoA substrates) was activated by K(+). 2. These properties were due to the presence, in different proportions in each tissue, of three classes of thiolase, all of which use acetoacetyl-CoA as substrate but which have different intracellular locations and substrate specificities and which differ also in kinetic and chromatographic behaviour. 3. Cytoplasmic thiolase activity was found to be widely distributed among different tissues and was due to an acetoacetyl-CoA-specific thiolase. This cytoplasmic activity was found to account for a significant proportion of the total tissue activity towards acetoacetyl-CoA in several tissues, and especially in the brain of newborn rats. 4. Mitochondrial thiolase activity towards acetoacetyl-CoA was due to two different classes of enzyme whose relative amounts varied with the tissue type. An oxoacyl-CoA thiolase of general specificity for the acyl-CoA substrate constituted one class, the other being a specific acetoacetyl-CoA thiolase that differed from its cytoplasmic counterpart in being greatly stimulated by K(+). 5. This activation by K(+) made it possible to calculate the tissue contents of mitochondrial acetoacetyl-CoA thiolase and mitochondrial oxoacyl-CoA thiolase from measurements of activity with acetoacetyl-CoA in tissue extracts under defined conditions. 6. The properties and the different thiolases and their tissue distribution is discussed with respect to their possible roles in metabolism.

Acetoacetates↗
PubMed1973

The acetoacetyl-coenzyme A thiolases of rat brain and their relative activities during postnatal development.

1. The apparent 3-oxoacyl-CoA thiolase activity of rat brain extracts is due to two different acetoacetyl-CoA thiolases, one cytoplasmic and the other mitochondrial. By the methods developed in the preceding paper (Middleton, 1973), the changes in activities of these two enzymes were determined during postnatal development. 2. Although the total brain acetoacetyl-CoA thiolase activity changes not more than 2-fold from birth to adulthood this masks large changes in the relative proportions of the two types of thiolase present. 3. Cytoplasmic acetoacetyl-CoA thiolase activity declines slowly from 4 units/g fresh wt. at birth to an adult value of 1.3 units/g fresh wt. 4. The mitochondrial acetoacetyl-CoA thiolase (activated by K(+)) rises rapidly in activity from 1 unit/g fresh wt. at birth to a peak value of 5 units/g fresh wt. at 20 days. After weaning the activity declines to 2.3 units/g fresh wt. in the adult. 5. These different developmental patterns are discussed in terms of the probable metabolic roles of the two brain acetoacetyl-CoA thiolases.

Acetoacetates↗
PubMed1972

The purification and some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from Baker's yeast.

1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (mumol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5mum, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10-30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.

Acylation↗
PubMed1972

The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast.

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have K(i) values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of K(i) with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).

Coenzyme A↗
PubMedDate not supplied

Dietary cyclandelate decreases pre-established atherosclerosis in the rabbit.

Rabbits were given a diet containing 1% (w/w) cholesterol for 7 weeks to initiate atherosclerosis and then transferred to a low-cholesterol diet (regression diet) with or without 0.5% (w/w) cyclandelate for 10 weeks. In those fed regression diet alone aortic atherosclerosis increased significantly. Cyclandelate treatment decreased the extent and severity of aortic atherosclerosis. Thus in rabbits receiving cyclandelate the total cholesterol and calcium content of aorta was decreased by 45% and 69% of control values, respectively. The effects of cyclandelate occurred without significant lowering of serum cholesterol or alteration of cholesterol content of liver or calcium content of serum, liver or muscle. The result suggests a specific action of cyclandelate on atheromatous accumulations of the aortic wall.

Animals↗