Electrophoretic characterization and subcellular distribution of hexokinase isoenzymes in red blood cells of rabbits

The isoenzyme pattern of hexokinase in rabbit red cells (erythrocytes, fetal erythrocytes and reticulocytes) were determined by means of agarose gel and disc electrophoresis. One duplicated hexokinase (4a and 4b according to the IUPAC-nomenclature) was detected in rabbit erythrocytes as also described for human erythrocytes. Besides the isoenzymes 4a and 4b reticulocytes also contain hexokinase 2 and 3 like rabbit and rat liver. The high KM glucose phosphorylating enzyme, hexokinase 1 could be demonstrated only under specific conditions in the reticulocytes during the initial stage of the anemia. After the fractionation of reticulocyte homogenates the total hexokinase activity was recovered in the mitochondria and cytosol to nearly equal amounts as revealed by the distribution of markers. Hexokinase 2 and 3 were detectable in reticulocytes and in isolated mitochondria only after the addition of certain dissociating agents. In contrast to the tightly bound mitochondrial hexokinases 2 and 3 the type 4a and 4b are more loosely bound and exhibit a bilocal distribution between mitochondria and cytosol of reticulocytes.     

Rat lung tissue is a site of alpha 1-proteinase inhibitor synthesis: evidence by cell-free translation

Poly(A) +RNA isolated from lungs of normal rats and of rats suffering from experimental inflammation was translated in a cell-free translation mixture from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-proteinase inhibitor and alpha 2-macroglobulin. Comparable levels of mRNA for alpha 1-proteinase inhibitor were found in rat lung tissue from control and experimentally inflamed animals. alpha 2-Macroglobulin mRNA could not be detected in rat lung tissue.     

Inhibition of protein synthesis by the β-subunit of spectrin

The 220 kDa β-subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme-regulated eIF-2 kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF-2 on its -subunit.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

Inhibition of peptide chain initiation in lysates from ATP-depleted cells. II. Studies on the mechanism of the lesion and its relation to similar alterations caused by oxidized glutathione and hemin deprivation.

The impairment of peptide chain initiation in lysates from ATP-depleted rabbit reticulocytes is accompanied by a loss of their ability to form the 40 S methionyl-tRNAfMet complex with a resultant failure to promote the AUG codon-dependent combination of the complex with the 60 S ribosomal subunit. These partial initiation reactions, as well as the overall protein-synthetic activity of the defective lysates could be restored by addition of a 0.5 M KC1 ribosomal extract or normal postribosomal supernatant or a 40-70% (NH4)2-SO4 fraction derived from it. Alternatively, reactivation of the impaired lysates could be achieved by supplementation with millimolar amounts of cyclic AMP or certain purine derivatives. The same subcellular fractions, as well as cyclic AMP or purine derivatives were also capable of overcoming the inhibition caused by incubating reticulocyte lysates in the presence of oxidized glutathione or in the absence of hemin. Severe intracellular ATP deprivation resulted in accumulation of a soluble translational inhibitor in the postribosomal fraction, thus resembling the parallel phenomenon described in hemin-deprived lysates. The striking similarities between the three kinds of inhibition studied by us point to an identical site of the underlying biochemical lesion, despite the different mechanisms mediating their induction.     

Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.     

Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.     

Protein synthesis in Drosophila melanogaster embryos. Two mechanisms for guanine nucleotide exchange on eukaryotic initiation factor 2

The mechanism for guanine nucleotide exchange with eukaryotic initiation factor-2 (eIF-2) from Drosophila melanogaster embryos was studied using the reaction eIF-2 X [3H]GDP + GDP (GTP) in equilibrium eIF-2 X GDP (GTP) + [3H]GDP. When highly purified eIF-2 is used the rate of nucleotide exchange is greatly reduced by Mg2+ and this reduction is overcome by the guanine-nucleotide-exchange factor (GEF) of rabbit reticulocytes. This GEF-dependent exchange is inhibited when Drosophila eIF-2 is either phosphorylated by the hemin-controlled inhibitor (HCI) of rabbit reticulocytes or treated with phosphatidylserine or a rabbit eIF-2 X phosphatidylserine complex. The Mg2+ impairment of guanine nucleotide exchange is less severe when highly purified eIF-2 is incubated at a higher temperature (37 degrees C) and is not observed at any temperature if partially purified eIF-2 is used instead of the highly purified factor. In the latter two cases the exchange is not inhibited by either phosphorylation with HCI or phospholipid treatment of Drosophila eIF-2, possibly suggesting that the observed exchange is not mediated by a GEF-like factor. Our data support two possible mechanisms for GDP/GTP exchange with Drosophila embryos eIF-2: a GEF-dependent exchange, similar to that described in rabbit reticulocytes, which may be regulated by phosphorylation of eIF-2, and a factor-independent exchange which appears to be insensitive to this type of control.     

High-molecular-mass proteinases in rabbit reticulocytes: the multicatalytic proteinase is an ATP-independent enzyme and ATP-activated proteolysis is in part associated with a cysteine proteinase complexed to alpha 1-macroglobulin

We have investigated the proteolytic degradation of [14C]methylcasein and 125I-labeled bovine serum albumin at pH 7.8 and 37 degrees C by lysates of rabbit reticulocytes purified from rabbit blood by two different procedures. (I) Lysates obtained from reticulocytes after removal of plasma and buffy coat as well as after washing of cells, degraded casein and albumin, and released from the two substrates 1.3%/h and 0.4%/h, respectively, of acid-soluble radioactivity. The activity towards both substrates was stimulated about 4-fold by ATP/Mg2+. Chromatography of whole blood on a column of cellulose prior to washing and lysis of cells had profound but differential effects on these activities in that stimulation of casein-degradation by ATP/Mg2+ was almost completely lost, whereas degradation of albumin, albeit at a low rate, was measurable in the presence of ATP/Mg2+ only. (II) Degradation of casein by these lysates is largely inhibited by a monospecific antibody against rabbit multicatalytic proteinase, whereas digestion of albumin is not affected by the antibody, either in the presence or absence of ATP/Mg2+. The latter activity is partially inhibited by a specific antibody against rabbit alpha 1-macroglobulin. (III) The immunoreactive amount of multicatalytic proteinase is about 1.2 micrograms per mg of lysate protein and almost identical in the two lysates. In contrast, the immunologically detectable levels of alpha 1-macroglobulin vary and are much lower in reticulocyte-lysates following chromatography on cellulose than in lysates from washed reticulocytes. (IV) Caseinolytic activity of multicatalytic proteinase, purified from rabbit reticulocyte lysate, is not activated by ATP/Mg2+ and the enzyme is proteolytically inactive towards albumin. On the other hand, a complex consisting of the proteinase inhibitor alpha 1-macroglobulin and the cysteine proteinase, cathepsin B, does degrade both substrates at pH 7.8, in an ATP/Mg2+-activated fashion. From these results it is concluded that the multicatalytic proteinase is an ATP-independent enzyme and a cellular constituent of rabbit reticulocytes whereas the activity stimulated by ATP/Mg2+ appears to be associated, at least in part, with a cysteine proteinase complexed to alpha 1-macroglobulin.     

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.     

Inhibition of exogenous RNA-dependent protein synthesis by a low-molecular-weight RNA from nuclear ribonucleoprotein particles of adenovirus-infected HeLa cells

Low-molecular-weight RNA (4S to > 5.5S) isolated from nuclear ribonucleo-protein particles of adenovirus-infected HeLa cells inhibited cell-free protein synthesis directed by polyribosomal RNA from rabbit reticulocytes by more than 80%. In a reconstituted system inhibitory RNA did not prevent the binding of Met-tRNA_f-GTP-IF ternary complex to 40S subunits; however, it repressed the formation of 80S from 40S-mRNA complex and 60S subunits. In binding assays in which authentic IF-M2A and IF-M2B were present, the inhibitor competed with messenger molecules for binding site(s) in IF-M2B. The inhibitory RNA appears to be a 5.5S RNA.     

Effect of normoxia and hypoxia on K(+) current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.     

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.     

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. β-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0–40% ammonium sulfate cut of intiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40–50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.     

Structural analysis of the messenger RNA cap-binding protein. Presence of phosphate, sulfhydryl, and disulfide groups

The messenger RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with [32P]orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with [1-14C]iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges. Species of CBP with faster or slower electrophoretic mobilities could be generated by treatment of the protein either with O2 in the presence of a catalyst or with dithiothreitol. The predominant form of the untreated protein migrated between these two forms.     

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.     

Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems

A highly efficient mammalian system was developed for the in vitro translation of exogenous rabbit globin messenger RNA. The system consists of purified ribosome subunits from mouse liver, rabbit reticulocytes, or guinea pig brain, partially purified initiation factors from rabbit reticulocytes, and elongation factors, termination factors, aminoacyl-tRNA synthethases and tRNA from rat liver in the form of pH 5-enzymes. (1) Emphasis was put on well-defined, structurally and functionally intact ribosomes, which we found to be the most difficult component of the system in terms of stability of activity. (2) An improved method for extraction of initiation factors from crude reticulocyte ribosomes was developed. Factor preparations of high specific activity were obtained by a simple, partial purification procedure. The crucial point was not to damage the ribosome structure during extraction of the initiation factors and to eliminate inhibitory components during extraction and purification. (3) The efficiency of the system was demonstrated quantitatively by showing that between $\text{3}\text{4}$ and one mRNA molecule per ribosome saturates the system and that each ribosome recycles over the mRNA several times. (4) Major uncertainties and ambiguities in the search for and identification of true initiation factors, as opposed to structural ribosome proteins needed for the reconstitution of damaged ribosomes, can be reduced by using this system.     

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.     

Effects of Ca2+ and lipoxygenase inhibitors on hexokinase degradation in rabbit reticulocytes

In rabbit reticulocytes more than half of the total hexokinase activity is mitochondrial bound and shows a fast decay during reticulocyte maturation. During in vitro incubation of rabbit reticulocytes, Ca²⁺ increases the decay of hexokinase while salicylhydroxamate (SHAM), an inhibitor of lipoxygenase, reduces the decay. Swelling of mitochondria, by incubation of the cells in hypotonic solutions, greatly enhances hexokinase decay, but both the Ca²⁺ and SHAM are still appreciable suggesting that Ca²⁺ and the swelling act by additive mechanisms, both able to influence hexokinase decay. This was confirmed by incubation of rabbit brain mitochondria in hypotonic solutions which does not promote any hexokinase decay, while the presence of Ca²⁺ does. Analyses of hexokinase isozymic pattern after incubation of reticulocytes in hypotonic solution both with and without Ca²⁺ and SHAM showed that the decay of hexokinase mainly involves the mitochrondrial bound isozymic forms.Abbreviations SHAM Salicylhydroxamate - HPLC High-Performance Liquid Chromatography