Lobster Tail Muscle Research Articles

Development and application of a high-performance liquid chromatography screening method for aromatic compounds in invertebrate tissues

Contamination of marine invertebrates by aromatic compounds (ACs) can occur from a variety of environmental sources, both natural and anthropogenic. Invertebrate species bioaccumulate ACs because they metabolize and eliminate them at a much slower rate than do vertebrates, such as fish. We have developed a high-performance liquid chromatography (HPLC) UV fluorescence screening method that measures ACs in invertebrate tissues. Screening methods have proven to be useful in response to environmental emergencies (e.g., oil spills) and in environmental monitoring because they are more rapid and less expensive than detailed analyses. This method was validated using three species of bivalves and lobster hepatopancreas and tail muscle as test samples. The AC screening method at naphthalene, phenanthrene, and benzo[a]pyrene wavelength pairs showed good correlation to GC/MS results. The method was also used successfully in response to the North Cape oil spill, off Narragansett, Rhode Island.http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p220.html

Immunoreactivities of p11 and calcyclin in baker's yeast, wheat germ and lobster.

After effectively eliminating the nonspecific cross-immunoreactivity with the affinity columns of anti-IgG agarose and IgG agarose, the potent immunoreactivities of p11 and calcyclin in wheat germ, lobster tail muscle, and three strains of baker's yeast were analyzed by Western blotting using mouse anti-p11 and rabbit anti-calcyclin. The occurrence of multiple bands may be due to either autolyses and/or the interactions between the p11 (or calcyclin) and other endogenous biological molecules. The results suggest not only a ubiquitous distribution and a universal Ca(2+)-mediating regulatory role of p11 and calcyclin in eukaryotes, but also an evolutionary conservation of these (S-100)-related proteins.

X-ray structure and catalytic mechanism of lobster enolase

Enolase prepared from lobster tail muscle yielded trigonal crystals with one 47 kDa subunit per asymmetric unit. X-ray data were collected on the apoenzyme at 2.4 A resolution and on a complex with Mn2+ and the inhibitor phosphoglycolate at 2.2 A resolution. The corresponding cDNA was amplified from a library of lobster muscle cDNA, and a sequence corresponding to residues 27-398 was determined. It is highly homologous to other enolases, including yeast enolase for which an X-ray structure is available. Yeast enolase was used as a starting point for crystallographic refinement, which led to models of lobster enolase having R-factors below 22% and good stereochemistry. These models are very similar to yeast enolase; they have the same fold with a beta 3 alpha 4 N-terminal domain followed by an atypical alpha/beta barrel. Lobster apoenolase and the ternary complex differ only in the position of three mobile loops. In the complex, a single Mn2+ ion is seen ligated to three carboxylates and three water molecules. Phosphoglycolate binds near, but not directly to, the metal. His 157, which belongs to one of the mobile loops, is in contact with the C2 atom of the ligand. A water molecule hydrogen-bonds to the carboxylate of the ligand and to those of Glu 166 and Glu 209. We suggest that His 157 is the base that abstracts the C2H proton, whereas the water molecule is part of a proton relay system keeping the substrate in the carboxylic acid form where the pKa of the C2H group is low enough for proton transfer to His 157. The resulting catalytic mechanism is different from those proposed on the basis of the yeast enzyme X-ray structures, but it fits with earlier biochemical and spectroscopic data.

A new crystal form of tropomyosin.

Tropomyosin crystals with a new morphology have been obtained from lobster tail muscle tropomyosin from which 11 residues at the carboxyl-terminus have been proteolytically removed to avoid head-to-tail polymerization. In contrast to the conventional Bailey crystal form in which the elongated tropomyosin molecules form a mesh, in the present crystals the molecules are packed side-to-side with the long axes parallel to the c-axis of the crystal. The unit cell is tetragonal with a = b = 109 A, c = 509 A, and the symmetry is either P4(1)2(1)2 or P4(3)2(1)2, with 4(1)(4(3)) helical axes parallel to the c-axis. This suggests that a group of molecules surrounding a local 4(1)(4(3)) axis is regarded as the building unit of the crystal. It is likely that the unit cell contains eight molecules with one molecule per asymmetric unit.

Determination of arsenobetaine, arsenocholine, and tetramethylarsonium cations in seafoods and human urine by high-performance liquid chromatography-thermochemical hydride generation-atomic absorption spectrometry

A simple method was developed for the determination of arsonium compounds in edible marine tissues (lobster tail muscle, peeled deveined shrimp, cod fillet, and cod liver oil) and human urine. The homogenized marine tissue (5-10 g (or an equivalent weight of freeze-dried powder)) was blended with methanol; the extracts were combined and flash evaporated. Alternatively, urine (5 mL) was diluted with 50 mL of ethanol and placed in a dry ice-acetone bath for 20 min. Supernatant was separated from the resulting precipitate by centrifugation and flash evaporated. Residues from either sample type were resuspended in water, filtered through an anion exchanger, and acidified. The arsonium analytes were partitioned into liquefied phenol, which was diluted with diethyl ether and back extracted with water. The combined water extracts were taken to dryness, redissolved in methanol, concentrated to 1 mL, and separated on a cyanopropyl bonded-phase column using a methanolic mobile phase containing 19% (v/v) diethyl ether, 1% (v/v) acetic acid, 0.12% (v/v) triethylamine, and pricrylsulfonic acid (0.200 g/L). Analytes were detected on-line by thermochemical hydride generation-atomic absorption spectrometry. Recoveries from tissues or from urine which had been spiked at 0.1-3.4 {mu}g of cation/g of fresh weight were 83% or greater from eachmore » of the five sample types.« less

Phosphorus-31 and proton NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with manganese(II) and cobalt(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)

Arsonium compounds in algae

Search for a precursor of the arsenobetaine discovered in Western Australian rock lobster tail muscle has led to an algal metabolite of radioarsenate having the properties of a trimethylarsoniumriboside derivative of the major arsenicals of aquatic plants, dimethylarsinoylribosylglycerol, its sulfate ester, and the corresponding riboside of phosphatidylglycerol. Such an arsonium compound could serve as metabolic precursor of arsenobetaine, the innocuous arsenical component of many marine food products. The oceanic diatom, Chaetoceros gracilis, cultured in radioarsenate produced a compound whose chemical, chromatographic, and electrophoretic properties are described. It was found to be identical to the trimethylarsonium derivative synthesized from the major algal arsenical, 1-(5'-dimethylarsinoyl-5'-deoxyribosyl)glycerol-3-O -sulfate.

Excitability and depolarization-release characteristics of excitatory nerve terminals in a tail muscle of spiny lobster.

In the deep abdominal L1-extensor muscle of the spiny lobster (Panulirus penicillatus) quantal excitatory postsynaptic currents (EPSCs) were recorded through macro-patch-clamp electrodes. Release of transmitter quanta from terminals was also elicited by depolarizing current pulses given through the recording electrode. The majority of terminals were excitable: on increasing the depolarization pulses, release was triggered at a threshold in an all-or-nothing manner. If excitation was blocked by tetrodotoxin (TTX), release was graded with depolarization reaching the amplitude of the all-or-nothing response at pulse amplitudes several times higher than the former threshold level. Some inexcitable terminals were also found: in these, release was graded for increasing depolarization pulses, and TTX did not alter the depolarization-release relation. Among the other types of terminals studied with the same technique, the proportion of excitable terminals in this lobster tail muscle is higher than in the crayfish opener and lower than in the frog's cutaneous pectoris muscle. The contribution of the increase in intraterminal Ca concentration to the control of release was estimated using facilitation of a test EPSC as an indicator of Ca inflow during a preceding depolarization pulse. This facilitation was found to have a maximum at a certain pulse amplitude, PF, and to decline for larger depolarizations. Release, however, rose considerably for depolarizations larger than those effected at PF. It is concluded that, like in crayfish and frog motor terminals, release is controlled directly by depolarization in addition to the control by Ca-inflow.

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase ( p-nitrophenyl phosphate as a substrate; Mg 2+ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.

Effect of modification of the 1-, 2-, and 6-positions of 9-β- d-ribofuranosylpurine cyclic 3′,5′-phosphate on the cyclic nucleotide specificity of adenosine cyclic 3′,5′-phosphate-and guanosine cyclic 3′,5′-phosphate-dependent protein kinases

A group of analogs of adenosine cyclic 3′,5′-phosphate (cAMP) and guanosine cyclic 3′,5′-phosphate (cGMP) with modifications in the 1-, 2- (or N 2), and 6 (or N 6)-positions of the purine ring were compared as activators of a cAMP-dependent protein kinase [PK(cAMP)] from bovine brain and of cGMP-dependent protein kinase [PK(cGMP)] from lobster tail muscle. The results suggest that the 6-amino group of cAMP is not required for the activation of PK(cAMP) by cAMP and that the 6-oxygen and 2-amino moieties of cGMP are required for the activation of PK(cAMP) by cAMP aIn the case of PK(cGMP) activation by cGMP, the 6-oxygen apparently accepts a proton from the enzyme and the 2-amino group apparently donates a proton to the enzyme.

A COMPARISON OF THE WATER HOLDING CAPACITY OF PRE- AND POST-RIGOR CHICKEN, TROUT AND LOBSTER MUSCLE IN THE PRESENCE OF POLYPHOSPHATES AND DIVALENT CATIONS

The water holding capacity (WHC) of rainbow trout (Salmo gairdneri) white muscle and lobster (Homarus americanus) tail muscle did not change from pre- to post-rigor. The trout muscle WHC values were similar to those of post-rigor chicken breast muscle and were not affected by the addition of sodium pyrophosphate (PPi), Mg, Ca or combinations of these. In contrast, the WHC of lobster muscle was like the WHC of pre-rigor chicken breast muscle. The pre-rigor lobster muscle showed a large increase in WHC values with the addition of pyrophosphate (205% of control) but with the further addition of Mg to the sample the increase was depressed (166% of control). Ca addition to the pyrophosphate sample even more markedly depressed the WHC (90% of control). In both cases, Mg and Ca seemed to have approximately the same effect on WHC whether PPi was present or not. Kena, on the other hand, increased the WHC of both pre- and post-rigor trout and lobster muscle. Ca seemed to negate the increasing effect Kena had on WHC. Kena plus Mg caused a large increase in the WHC of the prerigor lobster muscle; the WHC capacity with Kena alone was 123% of control while the WHC with Kena and Mg was 231% of control.

Catalytic properties of lactate dehydrogenase in Homarus americanus

Lobster tail and leg lactate dehydrogenases (LDH) have been characterized kinetically. The four binding sites for reduced coenzyme have been shown to be equivalent for the enzyme purified from lobster tail muscle. For the reduced form of 3-acetyl pyridineadenine dinucleotide, the K a = 1.4 × 10 7 M −1 S −1. The activity of the enzyme purified from the tail muscle is severely inhibited (90%) by high levels of pyruvate (10 m m) when assayed for pyruvate reductase activity at 11 °C; the reductase activity measured using the enzyme from the walking leg muscle was not inhibited by these high levels of pyruvate. Evidence is presented which indicates that the LDH from the tail muscle of the East Coast lobster forms an abortive ternary complex (enzyme-NAD +-pyruvate) which accounts for these inhibitory kinetics. The data suggest that the LDH from the tail muscles of the invertebrate lobster represents a “kinetic” heart-type l-specific LDH and that from the walking legs, a “kinetic” muscle-type l-specific LDH.

Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues.

A thermostable inhibition of ATP-protein phosphotransferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatography (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (Mr approximately or equal to 24,000) is specific for cAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985). The type II inhibitor (Mr approximately or equal to 15,000) competes for the enzyme with various substrate proteins (histone, alpha-casein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (cAMP- and cGMP-dependent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500-fold; this protein is thermostable, has acidic characteristics, and does not require Ca2+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.

Isolation of stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian heart devoid of inhibitory modulator of adenosine 3':5'-monophosphate-dependent protein kinase.

The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.

Isolation of stimulatory modulator from rat brain and its specific effect on guanosine 3′:5′-monophosphate-dependent protein kinase from cerebellum and other tissues

The stimulatory and the inhibitory activities of protein kinase modulator originally present in the crude modulator preparation obtained from rat brain was separated by Sephadex G-100 gel filteration. The isolated stimulatory modulator, as the crude modulator, augmented the activity of guanosine 3′:5′-monophosphate (cGMP)-dependent protein kinase from both the mammalian tissues (cerebellum, heart and lung) and the lobster tail muscle. The purified cGMP-dependent protein kinase was not activated by cGMP unless the stimulatory modulator, or the crude modulator, was added. The isolated inhibitory modulator, as the crude modulator, on the other hand, depressed the activity of adenosine 3′:5′-monophosphate (cAMP)-dependent protein kinase. It neither inhibited nor stimulated the activity of cGMP-dependent protein kinase.

A rapid and simple procedure for the determination of guanosine 3',5'-monophosphate by use of the protein-binding method.

A modification of the protein-binding assay for guanosine 3′,5′-monophosphate (Murad et al., 1971) is decribed yielding a substantial increase in sensitivity together with a more simplified procedure. The method employs a purified enzyme preparation from lobster tail muscle with a dissociation constant for cGMP of about 5 nM. The nucleotide-protein complex is separated from free cGMP by charcoal treatment. 0.05–0.1pmoles cGMP are detectable by this procedure. cGMP contents of seven discrete regions of rat brain were determined to show the usefullness of the described method.

LITERATURE ABSTRACTS

LITERATURE ABSTRACTS

Equilibrium binding of nicotinamide nucleotides to lactate dehydrogenases.

1. No discontinuities were observed during the continuous titration with NADH of the lactate dehydrogenases of ox muscle, pig heart, pig muscle, rabbit muscle, dogfish muscle or lobster tail muscle. The binding was monitored by either the enhanced fluorescence of bound NADH or the quenched fluorescence of the protein. A single macroscopic dissociation constant, independent of protein concentration, could be used to describe the binding to each enzyme, and there was no need to postulate the involvement of molecular relaxation effects. 2. The affinity for NADH decreases only threefold between pH6 and 8.5. Above pH9 the affinity decreases more rapidly with increasing pH and is consistent with a group of about pK9.5 facilitating binding. Muscle enzymes bind NADH more weakly than does the pig heart enzyme. 3. Increasing temperature and increasing concentrations of ethanol both weaken NADH binding. 4. NADH binding is weakened by increasing ionic strength. NaCl is more effective than similar ionic strengths derived from sodium phosphate or sodium pyrophosphate. 5. Commercial NAD(+) quenches the protein fluorescence of the heart and muscle isoenzymes. Highly purified NAD(+) does not, and its binding was monitored by competition for the NADH-binding sites. A single macroscopic dissociation constant is sufficient to describe NAD(+) binding at the concentrations tested. The dissociation constant is about 0.3mm and is not sensitive to changed ionic strength and to changed pH in the range pH6-8.5.

Protein Kinase Modulator from Lobster Tail Muscle: I. STIMULATORY AND INHIBITORY EFFECTS OF THE MODULATOR ON THE PHOSPHORYLATION OF SUBSTRATE PROTEINS BY GUANOSINE 3′,5′-MONOPHOSPHATE-DEPENDENT AND ADENOSINE 3′,5′-MONOPHOSPHATE-DEPENDENT PROTEIN KINASES

Abstract A heat-stable protein which modulates the activity of cyclic nucleotide-dependent protein kinases was purified from lobster tail muscle, and its properties were studied. The modulator is similar in several respects to the inhibitory factor reported by others (Appleman et al. (1969) Arch. Biochem. Biophys. 116, 39; Walsh et al. (1971) J. Biol. Chem. 246, 1977) to be present in rat and rabbit skeletal muscle. Polyacrylamide gel electrophoresis suggested the presence of aggregate forms of the modulator, each of which possessed both stimulatory and inhibitory activities. A minimum molecular weight of 34,000 for the modulator was estimated by using either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or amino acid analysis. The protein kinase modulator was shown to be a highly charged, acidic protein containing a low level of aromatic amino acid residues. p-Chloromercuribenzoate-treatment of the protein kinase modulator from lobster tail muscle caused a dramatic change in its actions on adenosine 3',5'-monophosphate (cyclic AMP)-dependent but not on guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase. Modulator activity was demonstrated in preparations from a wide variety of sources. However, the various preparations demonstrated considerable differences in the ratio of stimulatory to inhibitory activity. The modulator acts to alter the substrate specificity both of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, increasing the phosphorylation of some protein substrates and decreasing that of others. It is possible that the modulator may function in vivo by regulating the activity of cyclic GMP-dependent and cyclic AMP-dependent protein kinases through modification of their substrate specificity.

ATP synthesis in sarcoplasmic reticulum

Previous studies have reported that ATP is synthesized from ADP and P i in systems containing calcium phosphate-loaded sarcoplasmic reticulum membranes. We have repeated and confirmed this work in preparations from lobster tail muscle in which the only energy source was a calcium concentration gradient. This rules out possible contributions of high energy phosphate from other sources. We further found that FCCP, DNP, 0.1 m ammonium chloride or ammonium acetate, and several antibiotics including nigericin, valinomycin and gramicidin, had no effect on ATP-dependent inward calcium transport. Since these conditions increase membrane permeability to protons, potassium and sodium ions, or discharge pH gradients, there does not appear to be an obligatory coupling between calcium transport and gradients of other ions, as required by a chemiosmotically linked mechanism. This evidence is consistent with a transport mechanism in which inward transport is tightly coupled to phosphorylation of a transport enzyme by ATP. In the presence of a calcium concentration gradient of appropriate magnitude sarcoplasmic reticulum membranes catalyse synthesis of ATP by an apparent reversal of the inward transport reactions.