Differences in digestion and absorption of dietary protein in Atlantic salmon (Salmo salar) with genetically different trypsin isozymes

Digestion and absorption of dietary protein were studied through facilitation of amino acid in the plasma and white muscle after a single feeding. The comparison was made between Atlantic salmon with and without trypsin isozyme TRP-2*92. Higher absorption of dietary protein was associated with the presence of the isozyme, as the post-prandial total levels of free amino acids (FAA) in both plasma and white muscle were significantly higher in salmon with the isozyme than those in salmon without it. Higher digestion rate of the dietary protein in salmon carrying the isozyme was indicated by faster elevation of essential FAA in the plasma and of overall FAA in their white muscle. Other indications which suggest differences in nitrogen metabolism between salmon with and without the isozyme were the observations of significant differences in (a) the levels of lysine, hydroxyproline, alanine, aspartic acid, β-alanine, threonine, valine and a nitrogen-containing compound taurine in plasma, and (b) the levels of alanine, glutamic acid, glycine and anserine in white muscle.
Trypsin activity in the pyloric caeca showed less response to feeding than that in the intestine, but it may have consequence for growth as its activity was significantly higher in growing fish than in non-growing fish.     

Developmental changes in heart and muscle phosphofructokinase isozymes

Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel electrophoresis, and immunodiffusion with specific antisera. The results of these studies indicate that in skeletal muscle and heart the levels of the major liver phosphofructokinase isozyme (PFK-L2) and the muscle phosphofructokinase isozyme (PFK-M) are dependent on the developmental status of the rat. For example, PFK-L2 and PFK-M are present in fetal and early neonatal skeletal muscle; whereas in adult skeletal muscle, only PFK-M is detectable. By DEAE- cellulose chromatography, PFK-L2 activity was estimated to be 2.4 units/g (41% of total phosphofructokinase activity) in fetal muscle, very low and not resolved from PFK-M in 7-day neonatal muscle, and not detectable in adult muscle. Further, PFK-M activity was found to be 3.4 units/g (59% of total phosphofructokinase activity), 10 units/g, and 31.6 units/g in fetal, 7-day neonatal, and adult skeletal muscle, respectively. The developmental changes of heart phosphofructokinase isozymes differ considerably from that of the skeletal muscle phosphofructokinase isozymes. In fetal heart, PFK-L2 is the major phosphofructokinase isozyme (5.6 units/g), constituting 67% of total phosphofructokinase activity. Further, in fetal heart another phosphofructokinase isozyme (33% of total phosphofructokinase activity) was found by DEAE-cellulose chromatography which is different from PFK-M and PFK-L2. In 7-day neonatal and adult heart, PFK-M and PFK-L2 are the only detectable phosphofructokinase isozymes. Varying from 5.6 units/g (44% of total) in 7-day neonatal to 5.9 units/g (40% of total) in adult heart, PFK-L2 activity remains fairly constant. Also, PFK-M is very low in fetal heart but increases within 1 week postpartum to 5.5 units/g (50% of total activity) and to 8.9 units/g (60% of total activity) in adult heart.     

Distribution of AMP-deaminase isozymes in rat tissues

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Localization of lactate dehydrogenase isozymes in human muscle tissues by the mixed aggregation immunocytochemical technique.

Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A4 (M4) or LDH-B4 (H4) followed by the enzymes LDH-A4 and LDH-B4, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Insulin-like growth factor I binding and receptor kinase in red and white muscle

IGF-I receptors were partially purified from red and white skeletal muscle by lectin-affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I-IGF-I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF-I receptor preparation in the two fiber types was identical as determined by affinity cross-linking. The tyrosine kinase activity of the receptor from red muscle was 2-3-fold more active towards exogenous substrates in both the basal and ligand-activated states as compared to white muscle. These data show that there is IGF-I-dependent kinase activity intrinsic to IGF-I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF-I receptors in a parallel manner in vivo.     

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Comparative studies on the properties of the different isozymes

The properties of the isozymes of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) found in unfertilized frog egg have been compared to those found in adult tissues of Rana pipiens. Chromatographic, kinetic, and electrophoretic data indicate that, of the five electrophoretic forms found in egg, the isozyme with the least anodic mobility (isozyme I) is the same molecular species as the only isozyme found in heart, and the egg isozyme with the greatest anodic mobility (isozyme V) is identical to the major isozyme found in liver.The activity of egg isozyme I was markedly inhibited by the antibody to the skeletal muscle enzyme, which has been shown previously to cross-react with the cardiac enzyme, but was unaffected by the antibody to liver isozyme V; the opposite effects were observed with egg isozyme V. The antibody to the skeletal muscle enzyme inhibited egg isozymes II > III > IV whereas the antibody to the liver enzyme gave the reverse inhibitory pattern, e.g., isozyme IV > III > II.In vitro dissociation-reassociation of mixtures of isozyme I and V led to the formation of the other three isozymes. Similar experiments performed individually with either egg isozyme III or IV resulted in the production of predominantly isozymes III, II, and I due to the instability of isozyme V during the hybridization procedure.The above results indicate that isozymes I and V are tetramers of the respective parental subunits and that isozymes II, III, and IV are hybrid molecules with subunit assignments of (I₃V₁), I₂V₂), and (I₁V₃), respectively.     

Extra O2 consumption attributable to NADH2 during maximum lactate oxidation in the heart.

The lactate/pyruvate oxidation (Qo₂) ratio was 1.21 ± 0.04 for heart homogenates as compared to 0.92 ± 0.05 for white quadriceps muscle homogenates during state 3 respiration. The extra lactate Qo₂ could be accounted for by the oxidation of additional NADH₂ from lactate, assuming the oxidation of 12 H⁺/lactate and 10 H⁺/pyruvate. A high correlation of 0.92 was observed between extra lactate Qo₂ and activity of heart-type LDH isozyme. This finding and the mitochondrial location of heart-type isozyme (1) suggests the extra lactate Qo₂ in heart homogenates could represent the oxidation of NADH₂ formed from lactate by the mitochondria.     

Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.     

Comparative enzymology of AMP deaminase,adenylate kinase,and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15-to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution. Lower skeletal muscle activities in anurans may simply represent the contribution of tonic muscle fiber bundles containing low levels of adenosine monophosphate deaminase, but the explanation for the extremely high adenosine monophosphate deaminase levels in heart ventricular muscle is not apparent.Abbreviations AK adenylate kinase - AMP adenosine monophosphate - AMPD, AMP deaminase - CPK creatine (phospho)kinase - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl     

The functional coupling between MM isozyme of creatine phosphokinase (EC 2.7.3.2.) and MgATPase of myofibrils and (Na, K)ATPase of plasma membrane in heart cells]

The functional role of particulate MM isozyme of creatine phosphokinase (CPK) bound to heart myofibrils has been studied. It has been shown that in the presence of heart myofibrils and MgATP creatine phosphate can be used to rephosphorylate ADP formed in the MgATPase reaction. The rate of creatine phosphate splitting is determined by the kinetic properties of myofibrillar MgATPase and by the kinetic parameters of myofibrillar CPK. It has been found that a purified heart plasma membrane preparation contains high CPK activity. CPK isozyme bound to plasma membrane of heart cells is identical to MM isozyme of CPK and is able to rephosphorylate effectively ADP, formed in the (Na K)ATPase reaction. The rate of creatine phosphate splitting in these coupled reactions is sensitive to ouabain and is determined by the kinetic parameters both of the (Na, K)ATPase and plasma membrane CPK. The results obtained indicate the important role of myofibrillar and plasma membrane CPK in the intracellular energy transport processes.     

Flow cytometry of human spermatozoa

Summary Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A₄ (M₄) or LDH-B₄ (H₄) followed by the enzymes LDH-A₄ and LDH-B₄, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Molecular heterogeneity of rabbit heart phosphorylase kinase.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) from rabbit heart, when submitted to electrophoresis on Pevikon, separates into two discrete peaks A and B. The two peaks have been analyzed using reelectrophoresis, chromatography on DEAE-cellulose, thermal stability, inactivation by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) and reaction with an anti-muscle phosphorylase kinase antiserum. It can be concluded that rabbit heart extracts contain two isozymes of phosphorylase kinase. The more negatively charged isozyme seems to be identical with the muscle enzyme. The other isozyme resembles the liver enzyme but differs from the major fraction of the latter by its charge. It is likely that there exist at least three molecular types of phosphorylase kinase.     

Different isozymes of cytochrome c oxidase are expressed in bovine smooth muscle and skeletal or heart muscle

Cytochrome c oxidase (COX) was isolated from bovine smooth muscle (rumen), and compared with the enzyme from bovine liver, heart and skeletal muscle. A new isozyme of COX was found to be expressed in smooth muscle, which differs from the isozyme in liver and heart or skeletal muscle. SDS-PAGE as well as N-terminal amino acid sequencing of separated subunits from gel bands revealed the expression of the liver isoforms for subunits VIa and VIII and of the heart isoform for subunits VIIa in COX from smooth muscle.     

Physiological relevance of the changing subunit composition and regulatory properties of the 6-phosphofructo-1-kinase isozyme pools during heart and muscle development

During postnatal development, the subunit compositions of the 6-phosphofructo-l-kinase isozyme pools of heart and skeletal muscle are known to change. The isozyme pools from fetal muscle were composed of the L-type (60%), and M-type (36%) and C-type (4%) subunits and the isozymes from fetal and early neonatal heart contain nearly equal amounts of all three subunits. During postnatal development of both tissues, the proportion of the M-type subunit increases until it is the only type present in adult muscle and the major subunit in adult heart (7507o). The isozyme pool from fetal muscle exhibit a decreased affinity for fructose-6-P and a greater susceptibility to ATP inhibition compared to the M-rich isozymes which are subsequently present. The isozyme pools from fetal and early neonatal heart, if compared to the M-rich isozymes which are present later during heart development and to the fetal muscle isozymes, exhibited the least affinity for fructose-6-P and the greatest susceptibility to ATP inhibition. Comparison of the isozyme pools containing little or no C-type subunit with those from fetal and early neonatal heart clearly indicates that the presence of substantial levels of the C-type subunit imposed a decreased ability for fructose-2,6-P₂ to both lower affinity for fructose-6-P and antagonize sensitivity to ATP inhibition. Although still not thoroughly appreciated, it appears that the changing nature of the isozyme pools in these tissues permits regulation of glucose metabolism in a manner which allows efficient utilization of nutritional opportunities and which adequately meets the energy requirements of each tissue at different stages of development.Abbreviations PFK 6-phosphofructo-l-kinase - fructose-6-P D-fructose-6-phosphate - fr-t_ose-2,6-P₂ D-fructose-2,6-bisphosphate