Anisakis simplex: uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish

Adenosine triphosphatase activity stimulated by Mg2+ was greater in muscle mitochondria of fish infected with larval Anisakis simplex nematodes than in uninfected fish. When muscle mitochondria were isolated in a sucrose ethylene-glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid medium from fresh uninfected fish, they were loosely coupled, and their adenosine triphosphatase activity was comparable to that of mitochondria from rat tissue. Activity in infected fish was dose dependent, increasing with the number of worms per fish. Excretory secretory products or a cytoplasmic fraction of anisakines, when incubated with coupled rat mitochondria, also caused adenosine triphosphatase activity to increase. Storage of fish flesh caused an increase in adenosine triphosphatase activity, but such aging was not significant until 5 and 10 days after death in refrigerated and frozen samples, respectively. The Mg2+ stimulated adenosine triphosphatase activity of muscle mitochondria can be used to estimate the number of nematodes per market fish. The type of medium used to isolate the mitochondria is crucial in such studies; an ionic medium with Nagarse proteinase was optimal for fish muscle mitochondria.     

Sea urchin sperm guanylate cyclase antibody. Cross-reactivity various rat tissue guanylate cyclases.

After the repeated injection of sea urchin sperm guanylate cyclase into rabbits, antibodies to the enzyme were formed. These antibodies inhibited the particulate or the Triton-dispersed forms of the sperm enzyme by greater than 97%. The sperm adenylate cyclase, cyclic GMP phosphodiesterase, adenosine triphosphatase, guanosine triphosphatase, and 5'-nucleotidase enzymes were not affected by the antiserum. The antiserum inhibited the Triton-dispersed guanylate cyclase from rat heart, liver, lung, spleen, and kidney but did not inhibit the soluble form of the enzyme from any of these tissues. The inhibition of the Triton-dispersed enzyme in these tissues was partial, however, ranging from 30% (liver) to 70% (heart). These results provide evidence that adenylate cyclase is antigenically different from guanylate cyclase, and that the soluble form of guanylate cyclase is antigenically different from a particulate form of the enzyme in various rat tissues.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

The effects of univalent anions on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from the adrenal medulla

1. Influx and efflux of catecholamine and adenosine triphosphatase activity were studied in storage vesicles of bovine adrenal medulla. 2. In the absence of ATP the influx of catecholamine was slow and was not influenced by various anions, whereas the efflux increased in the sequence of anions given by the lyotrophic series. 3. In the presence of ATP the efflux was enhanced compared with that in the absence of ATP; the anion-dependent sequence, however, in which the efflux increased was the same as in the absence of ATP. 4. The ATP-dependent catecholamine influx and the adenosine triphosphatase activity are correlated. The sequence in which anions affect adenosine triphosphatase activity and catecholamine influx, however, is completely different from the lyotrophic anion series. 5. No correlation was found between adenosine triphosphatase activity and the efflux of catecholamine.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.     

Histochemical localization of lactate dehydrogenase isoenzymes in human skeletal muscle fibers.

Lactate dehydrogenase (LDH) activity was histochemically localized in fibers of the vastus lateralis muscle of men and for comparative purpose in the soleus and plantaris muscleo of rats. Human muscle fibers were identified as fast twitch (FT) or slow twitch (ST) from the histochemical stain for myofibrillar adenosine triphosphatase activity. Rat skeletal muscle fibers were classified as fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG), or slow-twitch-oxidative (SO) on the basis of NADH-diaphorase and myofibrillar adenosine triphosphatase activities. Heart-type (H) LDH was identified by inhibition of the muscle-type (M) isozyme with 4 M urea. Total LDH as estimated histochemically was highest in the human FT and rat FG fibers. This was predominantly the M-LDH isozyme. ST fibers of human and SO fibers of rat skeletal muscle had the least total LDH but the most H-LDH activity. The FOG fibers of rat muscle contained a total LDH activity intermediate to that of the FG and SO fibers and a combination of H- and M-LDH. There were no fibers in the human muscle samples studied that had LDH activities similar to the FOG fibers of rat muscle.     

Occurrence of dinucleosidetriphosphatase in the cytosol and particulate fractions from rat liver

Dinucleosidetriphosphatase (EC 3.6.1.29) is present in both the 37,000 g rat liver supernatant and precipitate (50 mU/g each fraction). These two activities show matching molecular weights, isoelectric points, substrate specificities, Km values, bivalent cation requirements and inhibition by zinc (II). The particulate triphosphatase and a residual dinucleosidetetraphosphatase (EC 3.6.1.17) are solubilized by freeze-thawing or by Triton X-100. Detergent treatment also extracts an unspecific phosphodiesterase I activity (EC 3.1.4.1) which also splits dinucleoside polyphosphates. The above findings suggest the occurrence of cytosolic and particulate degradative pathways for dinucleoside polyphosphates.     

Isolation of subunits from trypsin-cleaved sarcoplasmic reticulum Ca2+ transport adenosine triphosphatase

Summary Controlled tryptic digestion of purified rat skeletal muscle sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-adenosine triphosphatase yields two products designated Fragments 3a and 3b with molecular weights of 65,000 and 56,000 respectively. The isolation of these products in high yield should facilitate exploration of the molecular characteristics of this adenosine triphosphatase. A simple, rapid method for accomplishing this isolation was developed which provides a high yield and utilizes mild conditions. The fragments obtained by this method were used to determine the phospholipid and sulfhydryl contents of Fragments 3a and 3b. In addition, information was obtained on the orientation of these adenosine triphosphatase components in the enzyme lipoprotein complex.The work was supported in part by Grant #1 P50 HL 19316 from the National Institute of Arthritis and Metabolic Diseases.     

Isolation of subunits from trypsin-cleaved sarcoplasmic reticulum Ca2+ transport adenosine triphosphatase.

Controlled tryptic digestion of purified rat skeletal muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-adenosine triphosphate yields two products designated Fragments 3a and 3b with molecular weights of 65,000 and 56,000 respectively. The isolation of these products in high yield should facilitate exploration of the molecular characteristics of this adenosine triphosphatase. A simple, rapid method for accomplishing this isolation was developed which provides a high yield and utilizes mild conditions. The fragments obtained by this method were used to determine the phospholipid and sulfhydryl contents of Fragments 3a and 3b. In addition, information was obtained on the orientation of these adenosine triphosphatase components in the enzyme lipoprotein complex.     

Characterization of a brain particulate bound form of creatine kinase

A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.     

The smooth muscle 132 kDa cyclic GMP-dependent protein kinase substrate is not myosin light chain kinase or caldesmon.

Atrial natriuretic peptide (ANP) stimulates the phosphorylation of three cyclic GMP-dependent protein kinase substrate proteins of 225, 132, and 11 kDa (P225, P132 and P11 respectively) in the particulate fraction of cultured rat aortic smooth muscle cells [Sarcevic, Brookes, Martin, Kemp & Robinson (1989) J. Biol. Chem. 264, 20648-20654]. Vrolix, Raeymaekers, Wuytack, Hofmann & Casteels [(1988) Biochem. J. 255, 855-863] have reported the presence of a 130 kDa cyclic GMP-dependent protein kinase substrate protein in the membrane fraction of pig aorta or stomach, and suggested that it may be myosin light chain kinase (MLCK). The aim of the present study was to determine whether P132 from rat aorta was MLCK or caldesmon. Although P132 co-migrates with purified chicken gizzard MLCK on SDS/polyacrylamide gels, it is distinct from rat aortic MLCK. Partially purified MLCK from rat aorta migrated as a 145 kDa protein on SDS/polyacrylamide gels. Immunoblotting the partially purified rat aortic MLCK with antibody to bovine tracheal MLCK identified rat aortic MLCK (145 kDa) and a corresponding 145 kDa protein in the particulate fraction of cultured rat aortic smooth muscle cells, but did not detect the 132 kDa protein. Phosphopeptide maps of purified rat aortic MLCK prepared by digestion with Staphylococcus aureus V8 protease were distinct from those of P132. P132 was not caldesmon, since antibodies to caldesmon cross-reacted with 136 and 76 kDa proteins in the particulate fraction of rat aortic cells, but not with P132. Furthermore, caldesmon was partially extracted from the particulate into the soluble fraction by heating at 90 degrees C, whereas P132 was not. These results demonstrate that the ANP-responsive cyclic GMP-dependent protein kinase substrate of 132 kDa from rat aortic smooth muscle cells is not MLCK or caldesmon.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

Histochemical properties of skeletal muscle fibers in streptozotocin-diabetic rats

Summary The response of rat gastrocnemius muscle fibers to chronic streptozotocin-diabetes was studied. Transverse sections of this muscle from normal and diabetic rats were histochemically assayed for reduced diphosphopyridine nucleotide-diaphorase, myofibrillar adenosine triphosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and alkaline phosphatase activities. Cross-sectional areas of the fiber types were measured, and fiber capillarization and populations estimated. Chemically-induced diabetes appeared to have little effect on the metabolic or morphological properties of slow-twitch fibers. However, a general dedifferentiation occurred in the 2 fast-twitch fiber populations. There was a loss of oxidative potential in the fast-twitch-oxidative-glycolytic fibers, and a significant decrease in size in the fast-twitch-glycolytic fibers. No change in the proportions of slow- and fast-twitch fibers in the muscles of diabetic rats occurred. It is concluded that hypoinsulinism has differential effects on the 3 fiber types in heterogeneous rat skeletal muscle, and that slow-twitch fibers are least affected by the diabetic condition.     

Studies on the intermediary carbohydrate metabolism of aquatic animals; the distribution of acid-soluble phosphorus and certain enzymes in dolphin tissues

1. Liver, kidney, brain, skeletal muscle, and cardiac muscle from one newborn and three adult long-snouted dolphins (Stenella plagiodon) were obtained for enzyme studies. 2. All of the dolphin tissues exhibited cytochrome oxidase, succinic dehydrogenase, and malic dehydrogenase activity. Considerable differences in the enzyme activities of the various tissues were noted, with cardiac muscle exhibiting the highest respiratory enzyme activity. The enzyme activities of dolphin tissues were lower than those of the corresponding rat tissues. 3. All of the dolphin tissues exhibited adenosine triphosphatase activity which was accelerated by magnesium and manganese but, in contrast to rat tissues, was only slightly activated by calcium. 4. Measurements of the distribution of acid-soluble phosphorus in dolphin tissues indicated that glycolysis in all of the tissues examined proceeded through the Emden-Meyerhof phosphorylation scheme. 5. The average glycogen content of dolphin skeletal muscle was 0.98 per cent as compared with 0.16 to 0.20 per cent for rat skeletal muscle. The high glycogen content of dolphin skeletal muscle indicates a ready source of substrate for glycolysis even during submergence when the blood supply may be differentially shunted to other organs. 6. Measurements of the organ weights of dolphins showed that the lungs occupy over three times and the liver one-half as much of the total body weight as do these organs in the rat. The heart and the thyroid gland of the dolphin are also larger in proportion to the total body weight than in the rat while the relative weights of the other tissues in the two species are about the same.     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Effect of thyroidectomy on fast and slow muscle fibres of rat gastrocnemius muscle

A combined histochemical, biochemical and electrophoretic study with respect to the enzymes succnic dehydrogenase(SDH), myofibrillar adenosine triphosphatase (m-ATPase), lactate dehydrogenase (LDH) isozymes and myosin light chains was carried out to investigate the response of rat gastrocnemius muscle (medial head). Twelve weeks after thyroidectomy, the results indicated a shift from fast to slow type pattern of LDH isozymes, fibre type transformation from Type II to Type I and a decrease in SDH and m-ATPase activity. The results suggest, possible thyroidal involvement in determining the phenotypic properties of skeletal muscle.     

Postnatal development of thiamine metabolism in rat skeletal muscle.

1. The activities of 2-oxoglutarate dehydrogenase, transketolase, thiamine pyrophosphokinase and thiamine triphosphatase and the concentrations of thiamine phosphates were almost the same between rat extensor digitorum longus and soleus muscles at 2 weeks of age. 2. These enzyme activities changed after 3 weeks of age in a different way depending on the muscle phenotype. 3. Thiamine diphosphate level and the activity of 2-oxoglutarate dehydrogenase increased only in soleus muscle and thiamine triphosphate level increased only in extensor digitorum longus during development.     

Tissue difference in cellular localization of thiamine phosphate esters

1. Thiamine phosphate levels were determined in the soluble and particulate fractions of various rat tissues. 2. There was marked tissue difference in the cellular localization of thiamine phosphates. 3. Brain thiamine triphosphate was localized only in the particulate fraction, whereas skeletal muscle thiamine triphosphate was in the soluble fraction as a protein-unbound form.     

Enzymic and nonenzymic mono ADP-ribosylation of proteins in skeletal muscle

The acceptors of endogenously catalyzed monoADP-ribosylation in the cell free extract from rat skeletal muscle was searched. The main acceptor proteins in particulate were electrophoretically 52, 80, 100, and greater than 200 kDa proteins in the presence of SDS, while that in cytosol were 36 and 39 kDa proteins. Although no ADP-ribosylation was observed in particulate when the substrate NAD+ was replaced by ADP-ribose, the same ADP-ribose adducts were also formed with higher degree in cytosol. These results indicate that an enzymic and nonenzymic monoADP-ribosylation occur separately in cytosol and particulate. One acceptor, 36 kDa protein, appears to be glyceraldehyde-3-phosphate dehydrogenase.