Raúl E. Trucco

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Abstract Studies were carried out to determine the Hill coefficients for the inhibition by F − of the erythrocyte membrane-bound Mg 2+ -ATPase, (Na + + K + )-ATPase and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of n for the inhibition by F − of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the (Na + + K + )-ATPase and acetylcholinesterase, whereas the Mg 2+ -ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of n for acetylcholinesterase shifted as predicted. These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.

The allosteric transitions from membrane-bound enzymes: behavior of erythrocyte acetylcholinesterase from fat-deficient rats.

Abstract The allosteric behavior of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from red cell ghosts of rats fed fat-sufficient and fat-deficient diets was investigated. Allosteric type kinetics with n = −1.6 have been obtained for the inhibition by F- in rats fed a fat-sufficient diet. In animals fed a fat -sufficient diet the values on n changed form −1.6 to −1.0. When these animals were then fed rats fed a fat-sufficient diet the values of n shifted from −1.0 to −1.6. This in vivo reversion was obtained after 8 days of refeeding. Two types of changes in the values of n were obtained in vitro in fat-deficient rats: (1) from −1.0 to −1.6 by solubilization of the membrane-bound enzyme with Triton X-100, (2) from −1.6 to −1.0 by resconstitution of the membrane-like structure from the soluble enzymatic preparation. The possibility that the structure of the membrane could be responsible for the changes in the phenomenon of phenotypic allosteric desensitization in the membrane-bound enzymes is discussed

Identification of two alkaline proteases and a trypsin inhibitor from muscle of white croaker (Micropogon opercularis)

Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE‐Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin‐like enzyme, and protease II those of a SH‐enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.

Seasonal variations in gonadosomatic index, liver-somatic index and myosin/actin ratio in actomyosin of mature hake (Merluccius hubbsi)

Abstract 1. 1. Seasonal variations in gonadosomatic index (GSI), in liver-index (LSI) and in myosin/actin ratio in actomyosin of mature hakes were investigated. 2. 2. A resting or slow recuperation period and another of rapid transformation (October-February) were observed in gonads of female hakes. The latter occurred earlier in male fish than in female ones. Spawning occurred in March. 3. 3. High LSI values were found after spawning and during gonadal resting period. Low hake LSI values with high GSI ones were observed. 4. 4. Myosin/actin ratio profile was very similar to LSI one. A linear correlation was obtained when LSI values were plotted against myosin/actin ratio ones ( r = 0.858, P

The effect of fat deprivation on the allosteric inhibition by fluoride of the (Mg2+)-ATPase and (Na+ + K+)-ATPase from rat erythrocytes

Abstract It has been found that the ATPases (ATP phosphohydrolase, EC 3.6.1.3) from rat erythrocytes are inhibited by F − . Allosteric type of kinetics with n = −2.1 for the (Mg 2+ )-ATPase and −2.8 for the (Na + + K + )-ATPase have been obtained for the inhibition by F − . In animals fed fat-deficient diet the value of n for the (Mg 2+ )-ATPase changed from −2.1 to −1.4 and for the (Na + + K + )-ATPase from −2.7 to −1.5. When these animals were then fed fat-sufficient diet the value of n increased to the normal values. The possibility that changes in the unsaturated fatty acid composition of the erythrocyte membrane were responsible for the changes in the value of n is discussed.

The convenience of the use of allosteric "probes" for the study of lipid--protein interactions in biological membranes: thermodynamic considerations.

Abstract One of the most commonly used methods to study enzyme (protein)-structure interactions at a more specific level is the use of the Arrhenius plots to detect the influence of the “environment” on the enzyme. We want to point out here that the use of a suitable “allosteric enzyme” would be a more sensitive method to detect influences of the environment than the study of Arrhenius plots. According to simple thermodynamic considerations, a change of more than 2.8 kcal/mol in the interaction between enzyme and membrane would be needed to give a noticeable change in the position of the break (Ti) of the corresponding Arrhenius plot. On the other hand, feeble changes in the membrane-enzyme interaction, of the order of 0·7–0·8 kcal/mol, would be enough to give a significant change in the values of n (Hill coefficient).

Fish muscle cytoskeletal network: Its spatial organization and its degradation by an endogenous serine proteinase

The extraction of white croaker skeletal myofibrils with KI rendered a residue in which a network of longitudinal and transverse filaments could be observed by scanning electron microscopy. A trypsin-like serine proteinase isolated from the same muscle was able to produce a complete and rapid disruption of the network, while major myofibrillar proteins were only slightly modified. This fact suggests that the disassembly of the cytoskeletal network may be an early event in the proteolysis of myofibrils. Desmin was not attacked by the proteinase under the assayed conditions, which indicates that some other unidentified component of the network would be the primary target of the action of the enzyme on myofibrils.

Action of a serine proteinase from fish skeletal muscle on myofibrils.

The action of a serine proteinase from fish skeletal muscle on myofibrils was studied. The enzyme was able to destroy the structural integrity of myofibrils, and to degrade both their major contractile and cytoskeletal constituent proteins. Proteolysis could be completely prevented by the addition of a trypsin inhibitor isolated from the same muscle.

Allosteric transitions and membrane-bound ATPase from rat tissues: The effect of fat deprivation on the allosteric inhibition by fluoride

Abstract In rats red a fat-sufficient diet, ATPases (ATP phosphohydrolase, EC 3.6.1.3) from heart, kidney and brain microsomes showed allosteric kinetics for the inhibition by F − , with values of n = −2.0 . In rats fed a far-free diet, the values of n for the ATPases changed from −2.0 to −1.0 in heart and kidney microsomes. When these animals were then fed a fat-sufficient diet the values of n reached the control values. In brain microsomal ATPases no modification of the values of n were found between both groups of animals. The regulatory properties of the membrane on bound ATPases are discussed.

Electron microscopical and biochemical studies of actomyosin from pre- and post-spawned hake

Abstract 1. 1. The ultrastructure of actomyosin obtained from pre- and post-spawned hake was investigated. 2. 2. A characteristic arrowhead structure was found on actomyosin filaments obtained from post-spawned hake. 3. 3. The protein obtained from pre-spawned fish showed a loss of the filamentous structure. 4. 4. The loss of the filamentous structure in actomyosin from pre-spawned hake was related to a decreased affinity between myosin and actin.