Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human crythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed the basis for a double isotopic differential labeling technique using [¹⁴C]- and [³H]FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins.

Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band.

Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane.

These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.     

Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells

Changes in the isoenzyme patterns and activities of the two enzymes creatine kinase (CPK) and fructose diphosphate aldolase have been followed during the course of differentiation of chick skeletal muscle cells in vitro. The characteristic isoenzyme transitions of both of these enzymes known to occur in developing muscle in situ can be demonstrated in extracts of cultured myogenic cells by cellulose polyacetate electrophoresis followed by specific enzymatic staining: MM-CPK replaces the embryonic BB-CPK, while aldolase isoenzymes containing A subunits replace the C-containing forms which predominate at earlier stages. The specific activities of both enzymes increase during in vitro differentiation. Although the major part of these concomitant changes occurs after myoblast fusion has reached a maximum level, analysis of their timing relative to the process of fusion indicates that the increases in the activities of both enzymes, as well as the accumulation of nuclei within myotubes, proceed exponentially from the beginning of the second day in culture. Fusion and enzyme accumulation are unaffected by addition of dibutyryl cyclic AMP (1 × 10^?4M) to the medium. In calcium-deficient medium, or in media containing 5-bromodeoxyuridine (BrdUrd) at concentrations from 0.2 to 7 × 10^?5M, fusion is almost completely blocked, while cell viability is maintained. The CPK and aldolase isoenzyme transitions fail to occur normally in both fusion-preventing media. This blockage of the normal differentiative changes is, however, less complete in the calcium-deficient cultures, which, in contrast to the BrdUrd containing cultures, contained a number of long bipolar cells thought to be able to differentiate without fusion. These results are interpreted as indicating that for most, but possibly not for all, myogenic cells in typical primary muscle cell cultures, fusion is a prerequisite for the parallel differentiative changes in CPK and aldolase isoenzymes. The possibility is discussed that a “cluster” of proteins, including CPK and aldolase, may be coordinately regulated during myogenesis.     

The use of lanthanum and cytochalasin B to study calcium effects on skeletal muscle differentiation in vitro

A dual effect of external Ca²⁺ on creatine kinase (CPK) accumulation during myogenesis has recently been demonstrated (Morris and Cole, '79). Ca²⁺ inhibits muscle-specific CPK accumulation at intermediate (50–100 μ) concentrations compared with both lower (no added Ca²⁺) and higher (2–3 μ) concentrations. Myoblast fusion, however, requires high Ca²⁺ and is inhibited at both low and intermediate Ca²⁺ levels. These effects are now investigated further by studying the effects of lanthanum ion (La³⁺), which interferes with Ca²⁺-binding to membranes and Ca²⁺-transport, and cytochalasin B, which affects the cell membrane and prevents cell fusion without inhibiting CPK accumulation. The results show that low concentrations (10–100 μ) of La³⁺ inhibit the appearance of the muscle-specific (MM) CPK isoenzyme during myogenesis without significantly affecting cell fusion or intracellular cyclic AMP levels. Three further observations are consistent with the existence of myotube-specific membrane-binding sites for Ca²⁺, which are involved in the stimulation of CPK accumulation on increasing external Ca²⁺ from intermediate to high concentrations. (1) CPK levels are not affected by La³⁺ at 0–50 μ external Ca²⁺. (2) CPK levels in cytochalasin B treated myoblasts are hardly affected by La³⁺ at any Ca²⁺ concentration. (3) In cytochalasin B treated cultures, CPK levels are not increased by raising external Ca²⁺ from intermediate to high levels. In contrast, the stimulation of CPK accumulation on decreasing external Ca²⁺ from intermediate to very low concentrations is not affected by either La³⁺ or cytochalasin B. Some alternative interpretations of the data are also considered, including direct disruption of a membrane Ca²⁺-binding site by cytochalasin B.     

Chemical Modification of Membranes : 1. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell

Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),¹ 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K⁺ leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.     

Pyruvate kinase in pig liver mitochondria

The existence of the pyruvate kinase (PK) in pig liver mitochondria was shown by monitoring photometrically the PK reaction in solubilised mitochondria with either phosphoenolpyruvate (PEP) or ADP used as a substrate. In distinction with the cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or ADP concentrations. The occurrence of the mitochondrial PK was confirmed by immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix. PEP addition to mitochondria resulted in reduction of the intramitochondrial NAD(P)⁺ inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of the pyruvate dehydrogenase complex. Citrate/oxaloacetate appearance outside mitochondria also occurred as result of PEP addition to PLM. Taken together these findings support a role for PEP itself in triggering fatty acid synthesis via its mitochondrial metabolism.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

Malic acid accumulation by Aspergillus flavus

Summary ¹³C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the ¹³C NMR showed that the ¹³C label from [1-¹³C] glucose was incorporated only to C-3 (-CH₂-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.     

Energy metabolism and contractile function of the heart in diabetic cardiomyopathy: effect of ischemia and reperfusion]

Physiological parameters, rates of mitochondrial respiration, high energy phosphate levels and creatine phosphokinase (CPK) activity were investigated in the hearts from control and alloxan-induced diabetic rabbits before and after 40-min total ischemia and reperfusion. Diabetic hearts demonstrated significant decreases in the rates of contraction (+dP/dt) and relaxation (-dP/dt), heart rates and cardiac work compared to control hearts. Determination of mitochondrial respiration rates in saponin-skinned fibers showed a low mitochondrial respiratory function in diabetic hearts. It was found that the ATP and ADP levels and the total and mitochondrial isoenzyme activities of CPK in diabetic hearts were lowered in comparison with control. A post-ischemic recovery of cardiac performance for diabetic hearts was better than in controls. After reperfusion diabetic hearts had increased ATP levels. The data obtained demonstrate some abnormalities of both cardiac performance and energy metabolism in the hearts of diabetic animals and a decreased sensitivity of the latter to ischemic injury.     

The effect of imidoesters,fluorodinitrobenzene and trinitrobenzenesulfonate on ion transport in human erythrocytes

Several amino-reactive chemical probes which differ in hydrophobicity and charge and in their ability to penetrate the red cell membrane were tested for their ability to modify K⁺ leak and inorganic phosphate (Pi) leak in intact human red cells. Methyl picolinimidate (MP), ethyl acetimidate (EA), methyl acetimidate (MA) are hydrophilic penetrating probes whereas isethionylacetimidate (IA) is a hydrophilic non-penetrating probe. The order of their effectiveness in inhibiting Pi leak was found to be MP>EA>MA>IA. This order is in decreasing hydrophobicity and suggests that some penetration into the bilayer or into hydrophobic domains of the anion transport protein is required to modify an amino group required for Pi permeability through the membrane. These imidoesters have little or no effect on K⁺ leak in the red cell.Trinitrobenzenesulfonate (TNBS) a relatively non-penetrating hydrophobic anionic probe and fluorodinitrobenzene (FDNB) a penetrating hydrophobic neutral probe have markedly different effects on K⁺ and Pi leak. TNBS has little effect on K⁺ leak but markedly inhibits Pi leak. The effect of TNBS on Pi leak is not blocked by prior treatment with IA suggesting that these probes sense different populations of amino groups in the membrane. FDNB nearly completely blocks Pi leak and markedly increases K⁺ leak. The results with TNBS and FDNB indicate an asymmetric arrangement of amino groups on the red cell membrane. Certain amino groups on the outer surface of the membrane regulate Pi permeability whereas certain amino groups on the inner surface of the membrane regulate K⁺ permeabilty. The data also suggest that these amino groups are in a hydrophobic domain.     

Developmental changes in creatine phosphokinase isoenzymes in neonatal mouse hearts.

The distribution of creatine phosphokinase isoenzymes differs in extracts of newborn and adult mouse hearts. Electrophoresis on acetate strips reveals the presence of BB, MB, and MM isoenzymes in the 2 day old neonate heart, with relative activities of 4%, 24% and 72% respectively. Beginning at 6 days of age, a fourth isoenzyme, shown to be associated with mitochondria, is seen moving toward the cathode. With age the distribution changes, with BB disappearing by 18 days. By 25 days the relative proportions of MB, MM and mitochondrial CPK have reached 5%, 86% and 9%, respectively, similar to the levels seen in the adult. The late appearance of the mitochondrial isoenzyme may reflect a difference in the requirement of the developing and adult heart for ATP and phosphocreatine.     

Stobadine and heart mitochondria

Previous work has shown, that stobadine-hydrochloride (-)cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-14-pyrido(4,3b) indole administered in a single dose 2 mg/kg of body weight reduces cardiotoxic effect of isoproterenol (1 mg/kg) as shown by lowered serum enzyme activities of AST, CPK, LDH and ALT. We studied the effect of stobadine in vivo on respiration, the level of ATP, malondialdehyde (MDA) and superoxiddismutase (SOD) in heart mitochondria. Serum enzyme activities of AST, CPK and LDH after stobadine application were significantly decreased. In mitochondria, respiration and activity of SOD were inhibited, level of MDA was increased and level of ATP was unchanged. The cardioprotective effect of stobadine is not linked to preservation of mitochondrial function. This effect is probably more complex and mediated on the level of the whole organism.     

Kinetics of [18F]1-fluoro-2,4-dinitrobenzene,a potential probe for the glutathione detoxification system,in perfused working rat heart

[¹⁸F]1-Fluoro-2,4-dinitrobenzene (FDNB), a substrate for the detoxification enzyme glutathione-S-transferase, was prepared by ¹⁸F-Cl exchange. It was administered as a rapid bolus in the perfused working rat heart and the kinetics of the label were followed by external coincidence detection and by analysis of the coronary effluents and tissue homogenates. The data suggest that part of the extracted tracer was released, part was dehalogenated and part was reversibly incorporated into a large tissue pool of FDNB that was not a substrate for defluorination. The rates of the specific processes, estimated from the residue data, may be sensitive to changes in the glutathione detoxification system and therefore applicable to investigation of the compromised myocardium.     

Creatine phosphokinase BB distribution in different structures of the human brain

Regional localization of creatine phosphokinase BB was studied in postmortem human brain and its stability was shown. The content of CPK BB in different brain structures was unequal: from 0.5 mcg/mg of protein in the occipital lobe and tuber cinereum to 4.5 mcg/mg in the frontal lobe. In the regional localization of CPK BB in the postmortem brain of schizophrenia patients, some changes in isoenzyme content were found as compared to the control group. The reduction of CPK BB concentration at schizophrenia was found in the frontal lobe (45%, P less than 0.001) and s. nigra (70%, P less than 0.001); the concentration was higher in the thalamus and occipital lobe (15%, P less than 0.001), as well as in the parietal lobe, cingulate gyrus, tuber cinereum, cerebellum cortex, inferior olive--50-80%, P less than 0.001.     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Ontogenetic Development of Creatine Phosphokinase in Skeletal Muscles and Heart from Pigs

Creatine Phosphokinase (CPK) in striated muscles shows only small changes in activity before birth. After birth and during the first month of extrauterine life the activity increases rapidly. The largest increase is seen in muscles with a glycolytic energy metabolism (m. long, dorsi) and the smallest in muscles with an oxydative energy metabolism (m. flexor dig. ped. sup.). The differences between these groups of muscles are statistically significant. In heart tissue the increase in CPK activity is lower, the levels amounting to 40 to 47 % of those in striated muscles. Early in fetal life only the BB isoenzyme is found in striated muscles. Synthesis of M subunits of GPK starts between day 76 and 65 before birth and increases rapidly after this time leading to disappearance of the BB isoenzyme 24 days prior to birth and of the MB isoenzyme at birth. In muscles with an oxydative as well as in muscles with a glycolytic metabolism all GPK activity after birth is caused by the MM isoenzyme. All three isoenzymes are present in heart tissue at the earliest prenatal stage investigated, the pattern being dominated by the BB isoenzyme. During further differentiation the MM isoenzyme increases and the BB isoenzyme decreases. The development is completed during the first month after birth with a final isoenzyme composition of 81 % MM and 19 % MB isoenzyme. kw|Keywords|k]pigs; k]ontogenesis; k]creatine phosphokinase; k]activity; k]isoenzymes     

Effect of lacidipine on ischaemic and reperfused isolated rabbit hearts

Lacidipine is a new developed dihydropyridine calcium-antagonist, showing a slow onset and long lasting-selective activity.To assess whether the administration of lacidipine protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit heart were infused with three different concentrations of lacidipine: 10^–10; 10^–9; 10^–8 M. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for CPK activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity, ATP production and calcium content and tissue concentration of ATP, creatine phosphate (CP) and calcium were determined. Occurrence of oxidative stress during ischaemia and reperfusion was also monitored in terms of tissue content and release of reduced (GSH) and oxidized (GSSG) glutatione. Treatment with lacidipine at 10^–10 and 10^–9 M had no effects on the hearts when perfused under aerobic condition, whilst the higher dose reduced developed pressure of 36%. The ischaemic-induced deterioration of mitochondrial function was attenuated. On reperfusion treated hearts recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced tissue and mitochondrial calcium overload, release of CPK and of noradrenaline and oxidative stress were also significantly reduced. The effects of lacidipine were dose-dependent. The lower concentration (10^–10 M) failed to modify ischaemic and reperfusion damage. The dose of 10^–9 M was cardioprotective, but the best effect was found at 10^–8 M.It is concluded that lacidipine infusion provides a dose dependent protection of the heart against ischaemia and reperfusion. Because this protection occurred also at 10^–9 M, in the absence of negative inotropic effect during normoxia and of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. From our data we can envisage two other major mechanism:-1) membrane protection-2) reduction of oxygen toxicity. The ATP sparing effect occurring at 10^–8 M is likely to be responsable for the further protection.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 μM) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) from 72 ± 5 to 3042 ± 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca²⁺]_i, the effect of LPC was examined in the Ca²⁺-free solution containing EGTA. Under the Ca²⁺ -free conditions, LPC did not increase [Ca²⁺]_i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca²⁺-present condition. Addition of ryanodine (10 μM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca²⁺-free and Ca²⁺ -present conditions. Preincubation of the myocytes with d-propranolol (50 μM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca²⁺ -free and Ca²⁺ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca²⁺]_i, and the Ca²⁺ -independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.