Heart-type Subunit Research Articles

Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

BackgroundIron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron.MethodsSequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA.ResultsDolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species.ConclusionThese results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

Regulation of energy transduction and electron transfer in cytochrome c oxidase by adenine nucleotides.

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H+/e- stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K(M) for cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H+/e- stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.

Sequencing of cDNA Clones that Encode Bovine Ferritin H and L Chains

The molecular weight of the liver-type subunit (L) of bovine ferritin is much larger than that of the heart-type subunit (H) as determined by SDS-PAGE (L, 20.5 kDa; H, 18.4 kDa). The migration of these two subunits on SDS-PAGE gels, relative to each other, is opposite to that reported for ferritin L and H subunits in other mammalian species (L, 19 kDa; H, 21 kDa). To determine the cause of this anomaly, full-length cDNA clones of the bovine L and H chains were isolated from a bovine spleen λ gt11 cDNA library and sequenced. The amino acid sequences of the L and H chains of bovine ferritin, deduced from their cDNA sequences, contained open reading frames coding for 174 and 180 amino acid residues with calculated molecular weights of 19,856 and 20,920 Da, respectively. The deduced amino acid sequence of the L chain shows 86%, 84%, 87%, 83% and 83% homology with the amino acid sequences of horse, human, rabbit, rat and mouse L chains, respectively. The H chain displays a higher homology with the human, rat and mouse H chains (91%, 92% and 93%, respectively). In addition, the bovine L chain did not contain the extra octapeptide present in rodent L chains, and bovine L and H chains did not react with concanavalin A. The bovine L and H chains expressed using a baculovirus expression system showed almost the same mobilities as those of bovine spleen ferritin, respectively, by SDS-PAGE. These results suggest that the much slower mobility of the bovine L chain compared with other mammalian L chains on SDS-PAGE cannot be attributed to insertion(s) of amino acid(s) or peptide(s) into the L chain, to the deletion(s) of them of it or to the addition of carbohydrate chain(s) but may result from significant differences in the binding affinity of SDS for bovine ferritin L chains.

Cytochrome‐c Oxidase in Developing Rat Heart Enzymic Properties and Amino‐terminal Sequences Suggest Identity of the Fetal Heart and the Adult Liver Isoform

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol-cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densitometrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223-231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.

Steady-state transcript levels of cytochrome c oxidase genes during human myogenesis indicate subunit switching of subunit VIa and co-expression of subunit VIIa isoforms

Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the β subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so-called liver-type form of subunit VIa of cytochrome c oxidase diminished during the course of differentiation, while transcription of the so-called heart-type form was induced. Transcripts for the liver-type form and for the heart-type form of subunit VIIa of cytochrome c oxidase were detected in all myogenic cultures; the levels of the heart-type form progressively increased during the course of differentiation. The levels of the other transcripts studied did not change substantially. The results suggest subunit switching of subunit VIa and co-expression of subunit VIIa isoforms during myogenesis. The differential changes in mRNA levels of the heart-type subunits VIa and VII a and the differential changes in mRNA levels of the liver-type subunits VIa and VIIa demonstrate that different transcriptional regulation mechanisms are present for both heart-type genes as well as for both liver-type genes.

Lactate dehydrogenase subunit activities in hearts of aging rats

Diets with 40% of calories as fat with poly-unsaturated to saturated ratios of either 0.2 beef tallow (BT) diet or 1.0 mix (M) diet were fed to rats of both sexes. Groups were killed at 9, 12, 15, 18, and 21 mo. of age. To estimate the effects of age, sex and dietary fat on lactate dehydrogenase (LDH), activities of muscle type and heart type subunits were assayed biochemically in heart homogenates using differential substrate concentrations of 1.0 × 10−2 M and 3 × 10−4 M pyruvate, respectively. For comparison of methods, LDH activity was also determined histochemically in frozen sections of hearts, using lactate as a substrate.

A study of binding-solubilization of some glycolytic enzymes in striated muscle in situ.

The solubilization of lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and alpha-glycerophosphate dehydrogenase (GAPDH) was studied in pressed muscle as a function of ionic strength and NADH concentration. The results indicate that these factors affect the binding-solubilization of LDH and GAPDH in a similar way to their effect in dilute homogenized tissue. Alpha-glycerophosphate dehydrogenase was included as a typical soluble enzyme, since we have been unable to demonstrate its binding to subcellular fractions under any conditions. As with homogenized tissue, LDH was less susceptible to solubilization by ionic strength than GAPDH. It was demonstrated that LDH isozymes richer in heart-type subunits were more easily removed from muscle by centrifugation-imbibition than isozymes richer in the muscle-type subunits. This was interpreted as indicating that binding of the enzyme to subcellular structures was a major factor in the restricted removal of these enzymes from muscle, since only the muscle-type subunit is capable of binding to subcellular particles. It was further demonstrated that LDH could be taken up into muscle tissue, depleted in the enzyme, against an apparent concentration gradient. This was also interpreted as binding of the enzyme to the particulate structure of the muscle. Furthermore, this uptake of LDH occurred under conditions of ionic strength (0.25) and pH (7.5) that would prevent binding of the enzyme to the particulate fraction of a dilute suspension of homogenized muscle tissue. Thus, physiological conditions of pH and ionic strength do not necessarily induce solubilization of chicken breast muscle LDH in situ. Data obtained with dilute tissue homogenates, therefore, may not necessarily be easily and safely extrapolated to conditions in situ.

Automated kinetic determination of lactate dehydrogenase isoenzymes in serum.

A steady-state kinetic method has been revised for measuring lactate dehydrogenase isoenzyme activities, which relates the inhibition of heart-type isoenzyme activity to the overall isoenzyme composition of the enzyme subunits. The method depends on the pH-dependent formation of an inhibitory ternary complex by the heart-type isoenzyme with NAD+ and pyruvate (if the reaction is measured by NADH oxidation). A preincubation step in the previous method is eliminated. The isoenzymes are measured by measuring the reduction of pyruvate in two different concentrations, which favor either the total or fractional activity, depending on the concentrations of pyruvate and the percentage of heart-type subunits. The method has been adapted to a centrifugal analyzer, which has speeded automated isoenzyme determinations, with an accuracy comparable to that for electrophoretic methods.

Lactate dehydrogenase isoenzyme pattern in the eye muscles. Deviation in myopia.

The lactate dehydrogenase isoenzyme patterns in 46 extraocular eye muscle samples removed at surgery for squint were determined by acrylamide-gel electrophoresis and reduction of NAD coupled with formazan reaction. Muscle type subunits predominated in the isoenzymes of the medial and lateral rectus muscles of emmetropic and hypermetropic eyes, whereas heart type subunits predominated in those of myopic eyes.

Properties of water-insoluble matrix-bound lactate dehydrogenase

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K m values for its substrates. K m values for pyruvate and lactate were 8 × 10 −5 m and 4 × 10 −3 m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H 4) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M 4) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.

Lactate dehydrogenase: Electrophoretic behaviour electron microscopy and structure

The covalent binding of GSH and cysteine to mouse lactate dehydrogenase by disulphide bonds, gives rise to all but five bands of the complex electrophoretograms of the enzyme [Dudman, N.P.B. (1969) Biochem. Biophys. Res. Commun., 36, 608]. Other explanations have been proposed for this complexity, including that the enzyme exists as a number of conformational isomers [Houssais, J.-F. (1966) Biochim. Biophys. Acta 128, 239], and that the enzyme arises from the expression of three different genes [Costello, L. A. and Kaplan, N. O. (1963) Biochim. Biophys. Acta 73, 658]. The basis for these proposals is examined critically. From electrophoretograms of mouse lactate dehydrogenase both fully reduced, and covalently bound to GSH and cysteine, it is concluded that each muscle-type subunit has one especially active -SH group, but that the heart-type subunit has none. The state of ionization of the covalently bound GSH and cysteine residues, and of the free enzymatic thiol residues is also derived. Mouse lactate dehydrogenase is found to be adsorbed by some batches of polyacrylamide. Cathodic sub-bands of Isoenzyme 5 are bound most firmly, and adsorption appears strongest when the enzyme is fully reduced. Acrylamide gels containing pyruvate and NADH appear not to adsorb the enzyme. The possible effects of differing geometrical arrangements of subunits upon the electrophoretic pattern of lactate dehydrogenase are considered. Subunits of the enzyme from beef heart are found by electron microscopy to be arranged in flattened tetrahedra. This observation, taken with the electrophoretic pattern of the enzyme, indicates that beef lactate dehydrogenase comprises subunits of at least four distinguishable types.

Lactate Dehydrogenase Isozymes in Longnose and Blacknose Dace (Rhinichthys cataractae and R. atratulus) and Their Hybrid

Electrophoresis of tissue extracts of both longnose and blacknose dace reveals five isozymes corresponding to tetramers of heart and muscle type subunits. The species are readily distinguished by the greater electrophoretic mobility of longnose dace isozymes that contain heart type subunits. The F1 hybrid displays the multiple band pattern expected in a fish able to produce two kinds of heart subunit. In addition a variant type of muscle lactate dehydrogenase has been detected in the longnose dace.