Adenosine deaminase in the adductor muscle of the scallop, Patinopecten yessoensis

1. The scallop enzyme was separated by DE52 ion-exchange chromatography into two forms with the same mol. wt of 38,000 and similar characteristics. 2. The enzyme was inactivated in the absence of dithiothreitol and complete reactivation was achieved by adding the agent within a critical storage period. 3. The apparent values of pKm and Vmax sensitively increased as ionic strength was raised to 250 mM and phosphate and sodium ions elevated the former value with a further increase of the ionic strength. 4. The apparent activation energies for the alpha (Vmax/Km) and beta (Vmax) parameters of both the forms were approximately 5 and 8 kcal/mol, respectively. 5. The enzyme deaminated 2'-, 3'-deoxyadenosine and 2',3'-isopropylidene adenosine but did not deaminate 5'-deoxyadenosine, alpha-adenosine and adenine nucleotides. 6. The affinity for inosine was much lowered with a high Ki value. Adenine and purine riboside inhibited the enzyme completely, and coformycin was a tight, slow binding inhibitor.     

Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle.

1. A Ca(2+)-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of Mr approximately 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18 degrees C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.     

虾夷扇贝的多态性微卫星座位

虾夷扇贝(Patinopectenyessoensis),为冷水性贝类,是世界最重要的养殖经济贝类之一。原产于俄罗斯千岛群岛的南部水域,日本北海道及本洲北部。中国80年代初从日本引种,并开始养殖,目前已经在黄海北部形成规模化和产业化养殖,其中以大连长海养殖规模最大,近10年来创造出了数十亿     

Substrate specificity of aminopeptidase from the mid-gut gland of the scallop (Patinopecten yessoensis)

An action for various peptides and a kinetic study for amino acid p-nitroanilides (pNAs) and 4-methylcoumaryl-7-amides (MCAs) were performed with purified aminopeptidase from the mid-gut of the scallop. The enzyme preferred dipeptides having Ala, Met, and Phe in the amino-terminal or the penultimate position from the amino-termini. The catalytic efficiencies, k(cat)/K(m) values for Ala-pNA and MCA were the highest in the tested substrates, and those for pNA and MCA substrates having Met or Phe were the next highest. The enzyme was found to be a new alanine-specific aminopeptidase.     

Marine sterols. VI(1). Sterols of the scallop, Patinopecten yessoensis.

The sterols of the scallop, Patinopecten yessoensis Jay, was found to contain over 20 components. The major components were delta5-sterols, and lesser amount of ring-saturated sterols were also present. Biogenetically unusual C26 sterols (24-norcholesta-5,22-dien-3beta-ol and 24-norcholest-22-en-3beta-ol) and 24(28)-cis-24-propylidenecholest-5-en-3beta-ol (29-methylisofucosterol), 22-trans-27-nor-(24S)-24-methylcholesta-5,22-dien-3beta-ol (occelasterol), and a new sterol, 22-trans-27-nor-(24S)-24-methylcholest-22-en-3beta-ol (patinosterol), were isolated and their structures were confirmed. Occurrence of 22-trans-(24S)-24-methylcholesta-5,22-dien-3beta-ol (24-epibrassicasterol) was confirmed. 22-cis-Cholesta-5,22-dien-3beta-ol was not found.     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

Purification of a protein kinase phosphorylating myosin regulatory light chain-a (RLC-a) from smooth muscle of scallop, Patinopecten yessoensis

A protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6-aminohexyl-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-kDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require calmodulin and is rather inhibited by CaCl2.     

Polymorphic microsatellite loci for population studies of the Japanese scallop,Patinopecten yessoensis

Fifteen microsatellite loci were developed to examine the population structure of Japanese scallop, Patinopecten yessoensis. These markers were tested in samples from two geographically distant populations: the Sakhalin Island (Russia) and Dalian City (China). The mean numbers of alleles, observed and expected heterozygosities between the two populations were summarized by locus, but none of them show significant divergence between the two populations. Most loci conformed to Hardy–Weinberg expectation with the exception of loci HLJX‐06, HLJX‐12, HLJX‐13 and HLJX‐28, which had heterozygote deficits.     

Monoclonal antibodies toward scallop (Patinopecten yessoensis) testis and wheat germ calmodulins.

A monoclonal antibody (IM7) toward scallop testis calmodulin and another one (PBE2) toward wheat germ calmodulin were produced. Ca2+ was required for IM7 to react with scallop calmodulin. IM7 reacted with the C-terminal region (Asp78-Lys148) of the calmodulin. As observed on competitive ELISA, IM7 reacted with chicken calmodulin, but not with Euglena gracilis or wheat calmodulin, troponin C, myosin light chains, or parvalbumin. It is assumed that the cluster of Thr143, Thr146, and Ser147 in the C-terminal region acts as the antigenic site. IM7 (and Fab of IM7) inhibited the activities of myosin light chain kinase and cAMP-phosphodiesterase. PBE2 reacted with wheat germ calmodulin irrespective of the presence or absence of Ca2+, the antigenic site being in the N-terminal region (Ala1-Met37). It reacted with wheat and spinach calmodulins, but not with scallop, chicken, or Euglena calmodulin, troponin C, myosin light chains, or parvalbumin. PBE2 had no effect on the activities of myosin light chain kinase and cAMP-phosphodiesterase.     

Vitellogenin synthesis in the ovary of scallop,Patinopecten yessoensis: Control by estradiol-17 beta and the central nervous system

Elucidation of a profile of scallop vitellin formation associated with oogenesis and its endocrine control, and identification of a vitellogenin synthesizing site were immunologically undertaken by using anti-scallop Vn serum. Vn content increased during ovarian growth and accounted for more than 80% of the water soluble protein of the ovary at the mature stage. In vivo injection of estradiol-17 beta (E(2)) resulted in an increase in Vn content in the ovary. In vitro accumulation of Vn in the ovarian tissue was promoted with E2 and a vitellogenesis promoting factor (VPF) from cerebral plus pedal ganglion which was heat stable, less than MW 10,000 and trypsin/chymotrypsin resistant. Estrogen receptor (ER)-like immunoreactivity was found in the growing oocyte and the auxiliary cell in close contact with growing oocytes, in which Vn immunoreactivity was also found. It is suggested that the vitellogenin synthesis occurred inside the ovary, especially in the auxiliary cell, and is controlled by E2 and VPF via ER.     

Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.     

Serotonin-like immunoreactivity in the central nervous system and gonad of the scallop,Patinopecten yessoensis

Summary The localization of neurons containing serotonin in the central nervous system and the gonad of the scallop, Patinopecten yessoensis, was examined immunohistochemically. In the central nervous system a large number of immunoreactive perikarya were observed in the following regions: a part of the anterior lobe of the cerebral ganglion; the posterior lobe of the cerebral ganglion; the pedal ganglion; and the accessory ganglion. No immunoreactive perikarya were found in the visceral ganglion. Numerous immunoreactive fibers were revealed in the neuropil of all central ganglia. In the gonadal region immunoreactive fibers were distributed around the gonoduct and along the germinal epithelium.This work was supported by a grant from the Ministry of Education, Science and Culture, Japan     

Effects of Pectenophilus ornatus (Copepoda) on the biomass of cultured Japanese scallop Patinopecten yessoensis.

There was a negative relationship between the number of parasitic copepods (Pectenophilus ornatus) and the dry weight condition index of the infected Japanese scallop (Patinopecten yessoensis) cultured in the Tsugaru Strait of northern Japan. The parasite is considered a serious pest of the commercial bivalve in cases of heavy infection.     

The levels of prostaglandins associated with the reproductive cycle of the scallop, Patinopecten yessoensis

The present experiment was undertaken to investigate the seasonal variations of levels of prostaglandins (PGs) and regulation of these levels in the ovary and hemolymph of the scallop. The levels of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the hemolymph and ovary increased during sexual maturation, and these levels in the ovary showed a marked increase in the spawning season. Consecutive administration of antiestrogen inhibited the increase of the levels of PGF2 alpha and PGE2 during sexual maturation. These results indicate that the seasonal variations of the levels of PGF2 alpha and PGE2 are closely related to the reproductive cycle, suggesting that PGF2 alpha and PGE2 may be involved in the sexual maturation and spawning of the scallop. Furthermore, it was supposed that estrogen likely plays a role in the regulation of PGs production in female, well known in mammals.     

Isolation of a cDNA encoding the motor domain of nonmuscle myosin which is specifically expressed in the mantle pallial cell layer of scallop (Patinopecten yessoensis)

It has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.     

Characterization of regulatory light chain-a myosin kinase from smooth muscle of scallop, Patinopecten yessoensis

The protein kinase that phosphorylates the regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was isolated from scallop smooth muscle (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163). The enzymatic properties of this kinase (aMK) were investigated using RLC-a as the substrate. The Km value for ATP was 6.5 microM in the presence of 27 microM RLC-a at pH 7.0, and that for RLC-a was 133 microM in the presence of 1 mM ATP. The Vm value at saturation of both RLC-a and ATP was 0.25 s-1 at pH 7.0. The pH activity curve for aMK was bell-shaped with a maximum at around pH 7.8. The aMK activity was inhibited strongly by an increase in the KCl concentration. aMK required Mg2+, but was inhibited by high concentrations of Mg2+. The optimum activity was seen at 3 mM MgCl2. The mode of inhibition of the aMK activity by Ca2+ was studied. Assuming that the binding of Ca2+ to aMK induces the inhibition, the dissociation constant of Ca2+ was estimated to be 64 microM. aMK also phosphorylated LC20 of chicken gizzard myosin at a similar rate to that for RLC-a and the DTNB light chain of rabbit skeletal muscle myosin at a more lower rate. The helix and beta-sheet contents of aMK were estimated to be 19 and 30%, respectively, from the CD spectrum.     

Regulatory light chain-a myosin kinase (aMK) catalyzes phosphorylation of smooth muscle myosin heavy chains of scallop, Patinopecten yessoensis

Regulatory light chain-a myosin kinase (aMK), which phosphorylates one of the myosin regulatory light chains, RLC-a, contained in the catch muscle of scallop, was also found to phosphorylate heavy chains of scallop myosin. After incubation of myosin isolated from the opaque portion of scallop smooth muscle (opaque myosin) with aMK in the presence of [gamma-32P]ATP, about 2 mol of 32P was incorporated per mol of the myosin. The radioactivity was mostly found in the heavy chain at 0.26 M KCl. The pH-activity curve and MgCl2 requirement for the heavy chain phosphorylation were similar to those for RLC-a phosphorylation. In contrast, the dependency of activity on KCl concentration was different from that for RLC-a. The heavy chain phosphorylation activity decreased with increase in KCl concentration up to 0.06 M, and then increased at concentrations over 0.06 M to a maximum at around 0.26 M KCl. This complicated profile probably reflects the solubility of myosin, and the phosphorylation site may be located in the rod portion insoluble at low KCl concentrations. Phosphorylation of heavy chain did not change the solubility of the opaque myosin molecule at all. The acto-opaque myosin ATPase activity in the presence of Ca2+ was found to be decreased to less than one-fourth by the heavy chain phosphorylation.     

Pancreatic lipase and pancreatic lipase-related protein 2, but not pancreatic lipase-related protein 1, hydrolyze retinyl palmitate in physiological conditions

The major sources of vitamin A in the human diet are retinyl esters (mainly retinyl palmitate) and provitamin A carotenoids. It has been shown that classical pancreatic lipase (PL) is involved in the luminal hydrolysis of retinyl palmitate (RP), but it is not known whether pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2), two other lipases recovered in the human pancreatic juice, are also involved. The aim of this study was to assess whether RP acts a substrate for these lipase-related proteins. Pure horse PL, horse PLRP2 and dog PLRP1 were incubated with RP solubilized in its physiological vehicles, i.e., triglyceride-rich lipid droplets, mixed micelles and vesicles. High performance liquid chromatography (HPLC) was used to assess RP hydrolysis by the free retinol released in the incubation medium. Incubation of RP-containing emulsions with horse PL and colipase resulted in RP hydrolysis (0.051+/-0.01 micromol/min/mg). This hydrolysis was abolished when colipase was not added to the medium. PLRP2 and PLRP1 were unable to hydrolyze RP solubilized in emulsions, regardless of whether colipase was added to the medium. PL hydrolyzed RP solubilized in mixed micelles as well (0.074+/-0.014 micromol/min/mg). Again, this hydrolysis was abolished in the absence of colipase. PLRP2 hydrolyzed RP solubilized in micelles but less efficiently than PL (0.023+/-0.005 micromol/min/mg). Colipase had no effect on this hydrolysis. PLRP1 was unable to hydrolyze RP solubilized in micelles, regardless of whether colipase was present or absent. Both PL and PLRP2 hydrolyzed RP solubilized in a vesicle rich-solution, and a synergic phenomenon between the two lipases was enlighten. Taken together, these results show that (1) PL hydrolyzes RP whether RP is solubilized in emulsions or in mixed micelles, (2) PLRP2 hydrolyzes RP only when RP is solubilized in mixed micelles, and (3) PLRP1 is unable to hydrolyze RP regardless of whether RP is solubilized in emulsions or in mixed micelles.