Muscle Isoenzyme Research Articles

Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKC zeta.

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: alpha, delta, epsilon, eta, and zeta isoenzymes were detected. PKC alpha associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKC delta required detergent treatment for extraction from the EDTA-EGTA-washed particulate fraction. PKC epsilon, eta, and zeta were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKC zeta, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKC zeta-immunoreactive protein (M(r) 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKC zeta with peptide epsilon (a synthetic peptide based on the pseudosubstrate domain of PKC epsilon) as substrate was 2.1 mumol P(i).min-1.(mg PKC zeta)-1 and, with peptide zeta as substrate, was 1.2 mumol P(i).min-1.(mg PKC zeta)-1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKC zeta (specific activity, 0.1-0.3 mumol P(i).min-1.mg-1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of alpha, beta, and gamma isoenzymes), were also poor substrates of PKC zeta (specific activities, 0.04 and 0.02 mumol P(i).min-1.mg-1, respectively). Chicken gizzard PKC zeta was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKC zeta are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.

Myocardial lactate dehydrogenase subunit ratio in cardiac allograft recipients

Allograft coronary artery disease (CAD) is a major long-term complication in heart transplanted patients. However, the metabolic basis of allograft CAD remains to be fully elucidated. We analyzed the lactate dehydrogenase heart (H) and muscle (M) isoenzyme pattern in endomyocardial biopsy specimens and the evolution of the H/M ratio to test whether changes in this ratio could be the earliest manifestation of allograft CAD. Twenty-four heart transplant recipients were followed up for 12 months. Endomyocardial biopsy was performed at 1, 2, 3, 6, and 12 months after transplantation. Lactate dehydrogenase 1 through 5 isoenzymes were separated by electrophoresis, and the H/M ratio was calculated. Two groups of patients were identified: group 1 (n = 20), patients without allograft CAD; and group 2 (n = 4), patients with poor outcome (three deaths, 1 case of low cardiac output) and angiographic and histologic evidence of allograft CAD. Both groups had similar H/M baseline values. The H/M ratio was higher (p = 0.01) in group 1 at 6 months (3.48 +/- 0.64 versus 2.17 +/- 0.43) and 12 months (3.76 +/- 0.92 versus 2.18 +/- 0.45) when compared with group 2. The H/M ratio increased from 2.78 +/- 0.89 at 1 month to 3.76 +/- 0.92 at 12 months (p = 0.02) in group 1 and decreased in group 2 (2.86 +/- 0.49 versus 2.18 +/- 0.45; not significant). Changes in H/M ratio reflect an anaerobic shift in the lactate dehydrogenase isoenzyme composition and can be taken as an early indicator of allograft CAD.

Differential modulation of expression of the two acylphosphatase isoenzymes by thyroid hormone.

The modulation of expression of the skeletal muscle and erythrocyte acylphosphatase isoenzymes by thyroid hormone has been investigated. Our results indicate a differential regulation of the two enzymic isoforms by tri-iodothyronine (T3) in K562 cells in culture: an increase in the specific mRNA during T3-stimulation is shown only for the skeletal muscle isoenzyme. A fast and transient T3 induction of the accumulation of the specific mRNA can be observed, reaching a maximum 8 h after hormone treatment and then rapidly decreasing almost to the steady-state level after 24 h. A nuclear run-on assay was performed to explore the mechanisms of this regulation. These studies indicate that T3 induction of skeletal muscle acylphosphatase mRNA is due, at least in part, to a fast and transient increase in the rate of gene transcription, within 4 h after hormone administration. A very rapid decrease is then observed within a further 2 h. T3-dependent accumulation of the mRNA for the skeletal muscle acylphosphatase requires ongoing protein synthesis, as confirmed by inhibition with cycloheximide or puromycin. These findings indicate that the transcriptional regulation of the gene may be indirect.

Creatine kinase isoenzyme profiles in the plasma of the domestic fowl ( Gallus domesticus): effects of acute heat stress

Creatine kinase isoenzyme activities in extracts of plasma, skeletal muscle, heart and brain tissue of domestic fowls were separated by anion exchange chromatography and tissue specific distributions of the isoenzyme designated MM-CK, BB-CK1 and BB-CK2 were demonstrated. The muscle isoenzyme ( MM-CK) was the predominant form in plasma (99 per cent) and its activity increased in response to an episode of acute heat stress.

Inhibitory effects of cadmium ion on extracellular Ca-independent contraction of rat aorta

Administration of clenbuterol to rats in the drinking water over a 4 day period increased incorporation of [3H]leucine into muscle protein and caused a slight reduction in urinary 3-methylhistidine but did not result in an increase in body or muscle weight. However, both urinary and liver taurine were significantly reduced at the highest dose of clenbuterol (2 mg · kg−1 · day−1). Serum creatine kinase, muscle isoenzyme (CK-MM) was raised and single muscle fibre injury was observed in the soleus muscle in animals treated with the middle dose (0.2 mg · kg−1 · day−1) and highest dose (2 mg · kg−1 · day−1). The reduction in the body pool of taurine caused by clenbuterol is of concern as taurine has been shown to have protective properties.

The biochemical effects of clenbuterol: with particular reference to taurine and muscle damage

Administration of clenbuterol to rats in the drinking water over a 4 day period increased incorporation of [ 3H]leucine into muscle protein and caused a slight reduction in urinary 3-methylhistidine but did not result in an increase in body or muscle weight. However, both urinary and liver taurine were significantly reduced at the highest dose of clenbuterol (2 mg · kg −1 · day −1). Serum creatine kinase, muscle isoenzyme (CK-MM) was raised and single muscle fibre injury was observed in the soleus muscle in animals treated with the middle dose (0.2 mg · kg −1 · day −1) and highest dose (2 mg · kg −1 · day −1). The reduction in the body pool of taurine caused by clenbuterol is of concern as taurine has been shown to have protective properties.

Phosphodiesterase isoenzymes in human ureteral smooth muscle: identification, characterization, and functional effects of various phosphodiesterase inhibitors in vitro.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide metabolism and, thus, contraction and relaxation of the muscle. At present, five different families of isoenzymes of PDE exist that show a distinct species-specific and organ-specific distribution. The aim of the present study was to analyze the PDE isoenzymes present in the human ureter and to evaluate the functional effects of isoenzyme-specific inhibitors in this tissue. Normal ureteral tissue was obtained during radical nephrectomies, homogenized, centrifuged, and the supernatant fraction was separated using DEAE-Sephacel anion-exchange chromatography. PDE assay was then performed and the isoenzymes characterized on the basis of their kinetic characteristics and their sensitivity to allosteric modulators and inhibitors. In vitro, longitudinal ureteral strips as well as ureteral rings were precontracted, and different selective and nonselective PDE inhibitors were added incrementally. Three different PDE isoenzymes were identified: PDE I (Ca/calmodulin-stimulated), PDE II (cyclic guanosine monophosphate-stimulated), and PDE IV (cyclic adenosine monophosphate-specific). All PDE inhibitors relaxed the strips dose-dependently with an EC50 of 30 microM for papaverine, 40 microM for zaprinast, 25 microM for quazinone, and 0.1 microM for rolipram. The existence of three different PDE isoenzymes was shown in this study. The ureter-relaxing effect of the PDE IV inhibitor at low concentrations, combined with its low effect on the systemic circulatory parameters, may open a possibility of using selective PDE IV inhibitors in the treatment of ureteral colics or ureteral stones.

Effect of Tubulin on the Activity of the Muscle Isoenzyme of Lactate-Dehydrogenase

The interaction between muscle-type lactate dehydrogenase (LDH m) and tubulin was investigated by monitoring the combined effect of NADH and tubulin on steady-state kinetics and the combined effect of NADH and pH on complex formation between tubulin and the enzyme. Steady-state kinetics showed that LDH m is inhibited by tubulin. Experiments with heart-type lactate dehydrogenase (LDH h) showed that the inhibition is unique to the muscle-type enzyme. The magnitude of the inhibition is dependent upon the concentration of NADH as well as the pH of the buffer medium. The enzyme was less sensitive to inhibition at 50μM NADH than at 10μM NADH. Since this effect of NADH is not due to an ionic strength contribution, it is deemed to be specific. In contrast to the absence of tubulin, its presence induced a modification of the kinetic behavior of LDH m; i.e., the velocity dependence on NADH concentration displayed a marked sigmoid response. The inhibition of LDH m by tubulin is more pronounced at lower pH values than at higher pH values. The pH-dependent inhibitory profile is shifted to the left (i.e., p K a is decreased) with increasing concentrations of NADH. This pattern is remarkably similar to that observed for the binding of the enzyme on Sepharose immobilized tubulin and is consistent with the premise that inhibition is a result of interaction between these proteins. NAD + was much less effective than NADH in dissociating LDH m from immobilized tubulin. Results from these in vitro studies are consistent with similar observations dealing with other glycolytic enzymes and cytoskeleton proteins, which show that enzyme catalytic properties are modified upon binding.

Studies on the muscle toxicant 2,3,5,6-tetramethyl p-phenylenediamine: effects on various biomarkers including urinary creatine and taurine.

The effect of the specific muscle toxicant, 2,3,5,6-tetramethyl p-phenylenediamine (TMPD), on urinary creatine and taurine, markers of testicular and liver dysfunction, respectively, has been investigated in male Sprague-Dawley rats. Damage to the gastrocnemius and soleus muscles was accompanied by a rise in serum creatine kinase (predominantly the muscle-specific isoenzyme, CK-MM), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Increases in serum alpha-hydroxybutyrate dehydrogenase (HBDH) and total lactate dehydrogenase (LDH) (mainly isoenzymes, LDH1 and LDH2), occurred but only minor damage to the heart and no rise in CK-MB, (heart muscle isoenzyme) was seen. Damage to stage XIV tubules in the testis was evident histologically after the highest dose. This was accompanied by an increase in LDH-C4 testis-specific isoenzyme and a decrease in serum testosterone. Apart from reduced serum albumin, no other serum parameters indicated liver damage and there was only slight liver steatosis in some animals at the highest dose. Urinary taurine was not significantly raised after any dose of TMPD, but there was a significant increase in urinary creatine after the highest dose. It can be concluded that in the presence of discrete muscle damage, the use of urinary taurine and urinary creatine as markers of liver and testicular dysfunction, respectively, is not confounded. However, a variety of different markers should be used in conjunction to fully delineate the tissue damage due to toxic chemicals.

Antisense peptides to the 43-57 region of acylphosphatase and to the 46-60 region of two isoenzymes of a low-M(r) phosphotyrosine protein phosphatase do not interact with the corresponding proteins.

Three peptides complementary to exposed regions of two low-M(r) phosphotyrosine protein phosphatase isoenzymes and of the acylphosphatase muscle isoenzyme have been synthesized. Each peptide was synthesized on two different types of resins; the peptides were anchored to the resins by amide linkages. The peptide resins were checked by amino acid analysis and Edman degradation and directly used for enzyme purification. Despite our attempts, none of the resins was able to bind significant amounts of the corresponding protein, indicating the lack of interaction between the three proteins and the corresponding complementary peptides. This result agrees with many other reports, confirming that the molecular-recognition theory has no general validity.

Cyclic nucleotide phosphodiesterase isoenzymes in guinea-pig tracheal muscle and bronchorelaxation by alkylxanthines

In this study the phosphodiesterase (PDE) isoenzymes in guinea-pig trachealis smooth muscle were separated by DEAE-Sepharose anion exchange chromatography, identified, and characterized. Furthermore the effect of theophylline and 1- n-butyl-3- n-propylxanthine (BPX) on the isolated PDE isoenzymes and on their tracheal relaxant effect were investigated and compared with the nonxanthine PDE inhibitors amrinone and Ro 20-1724. We identified five distinct isoenzymes in guinea-pig tracheal muscle; calcium/calmodulin-stimulated cyclic AMP PDE (PDE I), cyclic GMP-stimulated cyclic AMP PDE (PDE II), cyclic GMP-inhibited and amrinone-sensitive cyclic AMP PDE (PDE III), cyclic AMP-specific and Ro 20-1724-sensitive PDE (PDE IV), and cyclic GMP-specific PDE (PDE V). BPX strongly inhibited the PDE IV isoenzyme with high selectivity, while the inhibitory effect of theophylline was weak. The PDE IV inhibitors BPX and Ro 20-1724 synergistically increased the relaxant effect of the β 2-adrenoceptor agonist salbutamol in carbachol-contracted trachea much more strongly than theophylline. In contrast, amrinone, a PDE III inhibitor, hardly influenced the relaxant effect of salbutamol, suggesting that the PDE IV isoenzyme is functionally associated with β 2-adrenoceptors in guinea-pig trachea and that inhibition of this enzyme potentiates the ability of sulbutamol to increase the intracellular cyclic AMP content. These results indicate that the PDE IV isoenzyme plays a significant role in alkylxanthine-mediated relaxation of guinea-pig trachea.

Expression of lactic dehydrogenase isoenzymes in rabbit muscle during development

1. 1. Rabbit cDNA probes for H and M lactic dehydrogenase subunits were used to monitor mRNA levels in different muscle types during growth. 2. 2. At the same time, lactic dehydrogenase activity and relative quantities of H and M protein subunits were measured. 3. 3. The main results are that mRNA abundance depends on muscle type and age, and mRNA abundance is not correlated with enzymatic activity.

Thyrotoxicosis and Hungry Bone Syndrome—A cause of posttreatment hypocalcemia

We report a case of a 54 year old woman with a history of recurrent Graves' disease, treated previously by thyroidectomy and later by radioiodine, who subsequently presented with tetany. Laboratory results revealed a profound hypocalcemia (total calcium 1.00 mmol/L; ionized calcium 0.53 mmol/L) and hyperphosphatemia (2.66 mmol/L) with low levels of parathyroid hormone. Although the patient's symptoms resolved after 5 days of treatment, hypocalcemia and elevated serum levels of bone-specific' alkaline phosphatase (ALP, EC 3.1.1) activity and skeletal muscle isoenzyme (CK-MM) creatine kinase (EC 2.7.3.2) activity persisted to her discharge, 3 weeks later. Attention is drawn to the recognition and management of recalcification tetany due to the “Hungry (for calcium) Bone Syndrome,” a biochemical and hormonal disturbance of calcium homeostasis and bone metabolism in the posttherapy thyrotoxic patient with hypoparathyroidism. This condition can be monitored by the use of calcium profile investigations, including bone-specific ALP, in addition to routine laboratory tests of thyroid function.

Elevated serum creatine kinase (CK-MM) in petrol sniffers using leaded or unleaded fuel.

This study examined blood lead and creatine kinase levels in a group of 24 Australian Aboriginal males admitted to the hospital for treatment of severe petrol sniffing related illness after using only leaded petrol and 27 sniffers, 16 ex-sniffers and 13 non-sniffers from an isolated Aboriginal community using only unleaded petrol. Creatine kinase levels (which were nearly all creatine kinase-skeletal muscle isoenzyme indicating skeletal muscle damage) were correlated with blood lead levels and were elevated in active sniffers of leaded petrol on admission to hospital but were also increased in those sniffing unleaded petrol in the remote community. After fourteen days in hospital, median creatine kinase levels of leaded petrol sniffers dropped rapidly to levels similar to those of ex-sniffers and non-sniffers while median blood lead levels decreased but still remained higher than the other three groups. The data suggest that elevated creatine kinase associated with petrol sniffing may be due to compounds in petrol other than the lead additives, possibly volatile hydrocarbon components. Elevated creatine kinase may be useful in detecting current petrol sniffing activity, particularly in locations using unleaded petrol.

Bovine testis acylphosphatase: Purification and amino acid sequence

Two acylphosphatase molecular forms have been isolated from bovine testis. Their amino acid sequence was determined. One (ACY1) consists of 98 amino acid residues, while the other one (ACY2) consists of 100 amino acid residues. Both molecular forms are N-acetylated and differ only in the amino terminus. ACY2 has an additional Ser-Met tail with respect to ACY1. Both ACY1 and ACY2 are organ-common type isoenzymes and thus differ for about half of the amino acid positions from the previously sequenced bovine muscle isoenzyme.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification, kinetics and immunological properties.

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55,800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53,900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53,300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mumol.l-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Altering creatine kinase isoenzymes in transgenic mouse muscle by overexpression of the B subunit.

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. .The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells > more mature muscle cells > muscel. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control sujects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.

Effect of flosequinan upon isoenzymes of phosphodiesterase from guinea-pig cardiac and vascular smooth muscle

The effect of flosequinan and its sulphone metabolite BTS 53 554, on phosphodiesterase isoenzymes isolated from guinea-pig cardiac and vascular smooth using DEAE-cellulose chromatography was investigated. Zaprinast and milrinone showed peak I and peak III selectivity, and IBMX non-selective activity, against both cardiac and vascular smooth muscle isoenzymes, as expected for these reference inhibitors. Flosequinan and BTS53 554 demonstrated non-selective inhibition with similar potency against both cardiac and vascular smooth muscle isoenzymes and, overall, were the least potent compounds tested. The high inhibitory concentrations observed (IC 50 peak III 660 μM for cardiac tissue and 230 μM for vascular smooth muscle with flosequinan) relative to its clinically effective plasma concentration (10 μM) questions the relevance of phosphodiesterase inhibition to the efficacy of flosequinan in heart failure.

Isolation and sequence of a cDNA encoding human platelet phosphofructokinase

A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics).