Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding.

A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.

Alkaline phosphatase in human milk: a new heat-stable enzyme.

Alkaline phosphatase in human milk was found to be heat stable and have a molecular size of placental alkaline phosphatase, a Mr of 160,000. However, the milk alkaline phosphatase is different from placental alkaline phosphatase. The alkaline phosphatase from milk was endowed with higher surface charge and not inhibited by L-phenylalanine (2.5 mmol/l) and L-homoarginine (10 mmol/1). Both enzymes are sialylated.

Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda).

Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras.

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea:Decapoda).

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M(r) 70,000 and M(r) 67,000, as well as less intense bands of proteins with M, 64,000 and M(r) 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M(r) 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl2, CuCl2 and Pb(NH3)3 at millimolar concentrations, but less inhibition was observed with CaCl2.

Characterization of insulin receptor from the muscle of the shrimp Penaeus japonicus (Crustacea: Decapoda).

The beta-subunit of the insulin receptor from the muscle of the shrimp Penaeus japonicus exists as multiple subtypes with M(r) of 79,000, 77,000 and 75,000. Only the subunit of M(r) 79,000 is autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the human placenta. The detergent, Triton X-100, and the metal ion, Mn2+, caused a noticeable enhancement of the autophosphorylation of shrimp insulin receptors from the muscle. Okadaic acid activated the kinase activity of the insulin-stimulated insulin receptor, but not the basal activity of the insulin receptor without the addition of insulin. Further studies comparing the insulin binding of the shrimp insulin receptor in the regulation of kinase activity of the multiple beta-subunit subtypes from the shrimp muscle are under way.

The zeta protein kinase C isoform from the testis of the grey mullet Mugil cephalus with a specific reaction protein of M(r) 48,000 on oolemma.

The zeta protein kinase C isoform (PKC-zeta) was purified from the testis of the grey mullet Mugil cephalus and has relative masses (M(r)) of 65,000 and 63,000. The subunits of PKC-zeta from spermatozoa degenerated to M(r) 58,000 and 53,000 after continuous freezing and thawing. Proteins of M(r) 48,000 on the oolemma of the grey mullet Mugil cephalus were found to be the reaction proteins of the PKC-zeta from spermatozoa.

Interaction of integrin beta1 with cytokeratin 1 in neuroblastoma NMB7 cells.

Cytokeratin 1, an intermediate filament keratin, was isolated as a partner of the tyrosine kinase Src from neuroblastoma NMB7 cells. The cytokeratin 1-Src complex was found to be associated with the molecular scaffolder RACK1 (receptor for activated protein kinase C). Interestingly, the cytokeratin 1-Src-RACK1 complex was found to actively bind with membrane receptors such as integrin beta1. We are interested in using this complex to find downstream kinases and phosphatases that bind upon cytokine stimulation, especially during neurogenesis.

A heat-stable alkaline phosphatase from Penaeus japonicus Bate (Crustacea: Decapoda): a phosphatidylinositol-glycan anchored membrane protein.

1. A heat-stable alkaline phosphatase was purified from Penaeus japonicus, with a final specific activity of 21,280 U/mg of protein. 2. In polyacrylamide-gel electrophoresis under non-denaturing conditions, the purified shrimp alkaline phosphatase was found to have an identical molecular size and surface charge as the human placental enzyme. 3. By using SDS-PAGE, the monomers of shrimp alkaline phosphatase were discovered to have a Mr 55,000 but those of human placental enzyme with a Mr 70,000. Deglycosylation decreases the Mr values of the subunits to 33,000 for shrimp alkaline phosphatase. 4. The purified alkaline phosphatase from shrimp was recovered with both the attachment sites for sialic acids and phosphatidylinositol. 5. The shrimp alkaline phosphatase has an isoelectric point (pI) of 7.6 and the human placental enzyme has a pI of 4.8.

A neutral beta-galactosidase from the hepatopancreas of the shrimp Penaeus monodon (Crustacea: Decapoda): dimeric and sialyated.

1. A beta-galactosidase was purified ca 245-fold to homogeneity from Penaeus monodon, with a final spec. act. of 61.3 U/mg of protein. 2.By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp beta-galactosidase were discovered to have mol. wts of 31,000 and those of human placental enzyme, 32,000. Since the active shrimp beta-galactosidase was found to have a mol. wt of 66,000 by AcA 34 gel filtration chromatography,it was concluded that the purified shrimp enzyme was dimeric. 3.In contrast to the discovery of thermostability with human placental beta-galactosidase, the shrimp enzyme was found to be unstable to heating at 45 degree C for 10 min. Both enzyme activities were inhibited by Mn (2+) and Zn (2+) ions.4. The shrimp beta-galactosidase has an isoelectric point (pI) of 7.0, but the human placental enzyme has a pI of 5.5. Both enzymes were sialyated. 5.The shrimp beta-galactosidase has a pH optimum at 7.0 and its K(m) was 1.9 micrometer with 4-methylumbelliferyl-beta-D-galactoside as substrate. The human enzyme has pH optimum at 7.0 or 4.0, and its K(m) was 9.8 micrometer.

A comparative study of alkaline phosphatases among human placenta, bovine milk, hepatopancreases of shrimp Penaeus monodon (Crustacea: Decapoda) and clam Meretrix lusoria (Bivalvia: Veneidae): to obtain an alkaline phosphatase with improved characteristics as a reporter.

1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65 degrees C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.

Purification and properties of a beta-mannosidase from shrimp (Penaeus japonicus) hepatopancreas.

A beta-mannosidase was purified ca 720-fold to homogeneity from Penaeus japonicus, with a final spec. act. of 252 U/mg of protein. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp beta-mannosidase were discovered to have mol. wts of 31,000 and those of human placental enzyme have similar mol. wts. 3.The shrimp beta-mannosidase has an isoelectric point (pI) of 5.6 +/- 0.1, and the human placental enzyme has an identical pI. Both enzymes were sialyated. 4.The shrimp beta-mannosidase has a pH optimum at 5.0 and its K(m) was 123 micrometer with 4-methylumbelliferyl-beta-D-mannopyranoside as substrate. The human enzyme has pH optimum at 4.5 and its K(m) was 10 micrometer. 5.In contrast to the discovery of thermostability with human placental beta-mannosidase, the shrimp enzyme was found to be labile to heating at 45 degree C for 20 min. Both enzyme activities were inhibited by Hg(2+) and Cd(2+) ions. However, the shrimp enzyme is significantly more sensitive to the inhibition.

Characterization of phosphotyrosyl protein phosphatase fom the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda).

Phosphotyrosyl protein phosphatase, purified from the hepatopancreas of Panaeus japonicus, is a monomeric enzyme with a relative mass (Mr) of 28,000, as estimated by size-exclusion FPLC on a Superose 12. It has a hydrophobic domain and can be extracted with the detergent CHAPS.The specific activity of the purified enzyme was 9,800 units/mg of protein. The purified enzyme had an isoelectric point less than 4.6, and an optimal pH of 6.5 with either a synthetic peptide or autophosphorylated receptors for insulin as the substrate. The purified phosphotyrosyl protein phosphatase from shrimp hepatopancreas dephosphorylated human and shrimp receptors for insulin and was inactivated by ZnC1(2), LiC1, MgC1(2), and NaF.

Purification and some properties of alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda).

Alkaline phosphatase purified from the hepatopancreas of Penaeus japonicus is stable to heating at 65 degree C for 5 min. The specific activity of the purified enzyme is 25,000 units/mg of protein. After polyacrylamide gel electrophoresis under denaturing conditions, the purified alkaline phosphatase from shrimp was found to consist of deglycosylated monomers of Mr 40,000 and to retain the attachment sites for both sialic acid and phosphatidylinositol. The alkaline phosphatase from shrimp has an isoelectric point (PI) of 7.6 and becomes more alkaline after the removal of either sialic acid or phosphatidylinositol residues.

The binding of corticosterone to the class-theta glutathione S-transferase from the eyes of the shrimp Penaeus japonicus (Crustacea: Decapoda).

The anionic homodimeric theta glutathione S-transferase (EC 2.5.1.18), with specific activities of 4.6 units/mg with 1-chloro-2,4-dinitrobenzene as substrate, from the eyes of the shrimp Penaeus japonicus, binds to corticosterone, which was confirmed by the analysis of the conjugate by SDS-PAGE and fluorography of proteins with bound (3)H-corticosterone.The reaction of the theta glutathione S-transferase with corticosterone was prevented by the presence of glutathione at 10 mM.

GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphate but not mammalian GAP.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.

A sialidase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda): reversible binding with the acidic beta-galactosidase.

1. The sialidase purified from the hepatopancreas of Penaeus japonicus is able to bind the acidic beta-galactosidase in vitro. No protective protein, Mr 32,000, was detected in either purified enzyme preparation. 2. The specific activity of the isolated sialidase is 55.0 mU/mg of protein. After polyacrylamide gel electrophoresis under denaturing conditions, the purified shrimp enzyme was found to consist of monomers of Mr 32,000. 3. The sialidase from shrimp has an isoelectric point (pI) of 4.6 +/- 0.1. 4. The shrimp enzyme has the pH optimum at 5.0 and its Km was 5.5 microM with 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid as substrate. The enzyme activity was inhibited by either Hg2+ or Cu2+ ions.

Disrupting the geranylgeranylation at the C-termini of the shrimp Ras by depriving guanine nucleotide binding at the N-terminal.

In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian K(B)-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510-521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant S-Ras proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565-573) was unable to catalyze the transfer of [(3)H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441-449, 2000.

Purification of the delta isoenzyme of protein kinase C from the hepatopancreas of the shrimp Penaeus monodon with phosphorylation on tyrosine residues.

The delta isoenzyme of protein kinase C (PKC-delta), purified from the plasma membrane of the hepatopancreas of the shrimp Penaeus monodon is specifically phosphorylated at tyrosine residues, as demonstrated by specific dephosphorylation by phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. The specific activity of purified PKC-delta was 200 units/mg of protein. The subunits of M(r) 66,000, 62,000, and 58,000 of PKC-delta were not autophosphorylated after the addition of phosphatidylserine and diolein. However, the purified PKC-delta was active and catalyzed the phosphorylation of myelin basic protein. The kinase activity of the purified PKC-delta could be decreased after treatment with phosphotyrosyl protein phosphatase.

Carboxy-terminal CVLS-sequence-specific protein farnesyltransferase from the eyes of the shrimp Penaeus japonicus: purification and characterization.

Protein farnesyltransferase from the eyes of Penaeus japonicus farnesylates predominantly H-ras-specific carboxyl termini, with the sequence CVLS, but not the K-ras-specific sequence CVIM or the protein geranylgeranyltransferase-specific sequence CAIL. The purified protein farnesyltransferase from shrimp was found by immunoblotting and polyacrylamide gel electrophoresis under denaturing conditions to consist of subunits of Mr 49,000 and Mr 48,000. Since the active protein farnesyltransferase was found to have a relative mass of 100,000, the purified enzyme was deduced to be a heterodimer. The enzyme had an optimal pH of 6 and a K(m) of 14 +/- 1 microM with the synthetic peptide RTRCVLSH as the substrate. The enzyme was activated by Mn+2 and Mg+2 but inhibited by Ca+2 ions.