Epidermal secretions of terrestrial flatworms and slugs: Lehmannia valentiana mucus contains matrilin-like proteins

We describe the epidermal mucus of two types of terrestrial invertebrates: free-living flatworms (Tricladida: Terricola), and the slug Lehmannia valentiana (Gastropoda: Pulmonata). Both exhibited similar dry shear strengths (1.4-1.7 MPa). In denaturing gel electrophoresis, the protein fraction of flatworm mucus migrated mainly as a broad band (200-300 kDa). Slug mucus had a higher protein content than flatworm mucus but it contained more carbohydrate than protein, mainly as large heparan sulfate-like glycosaminoglycans. Proteins and glycosaminoglycans were both essential for the mechanical integrity of the slug hydrogel. The protein fraction of slug mucus contained approximately 12 larger proteins (30-300 kDa) and approximately 6 smaller ones (10-28 kDa). Complete cDNA clones were obtained for the slug mucus 40 kDa protein (Sm40; Genbank accession EF634345) and 85 kDa protein (Sm85; Genbank accession EF634346). Both proteins contain EGF-like repeats and von Willebrand A-domains, and therefore resemble vertebrate matrilins. Many of the larger slug mucus proteins appear to contain A-domains, and these may play a role in the unusual rheological properties of gastropod mucus.     

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain

Amino acid sequences were obtained for four peptides (p1, -2, -3 and 4) generated by chemical or proteolytic cleavage of a 25 kDa GTP-binding protein purified from human placental and platelet membranes. The peptides shared sequence similarities with those contained in several of the ras-related GTP-binding proteins. Peptide p2, a 12-mer, was homologous with a region of the GTP-binding proteins that contains a structural motif proposed to contribute to the nucleotide binding site. However, whereas nearly all GTP-binding proteins exhibit the residues NKXD as this motif, p2 contains TQID. Antisera (Ap1 and Ap3) raised against synthetic peptides corresponding to p1 and p3 specifically reacted on Western blots with the 25 kDa GTP-binding protein purified from human placenta, human platelet and bovine brain as well as with a 25 kDa polypeptide in various cell lines. These results demonstrate the widespread existence of an abundant 25 kDa GTP-binding protein which contains a putative nucleotide binding domain that is chemically distinct from that described for all GTP-binding proteins of known primary structure.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

Intestinal mucins: the binding sites forsalmonella typhimurium

Mucus-bacterial interactions in the gastrointestinal tract and their impact on subsequent enteric infections are poorly delineated. In the present study, we have examined the binding ofSalmonella typhimurium to rat intestinal mucus and characterized a mucus protein (Mucus-Rs) which specifically binds to S. typhimurium. Both virulent (1402/84), and avirulent (SF 1835) S. typhimurium were observed to bind to crude mucus, however, the virulent strain showed 6 fold more binding as compared to avirulent strain. Fractionation of crude mucus on sepharose CL-6B resolved it into three major peaks. Maximal bacterial binding was observed with a high mol. wt. glycoprotein corresponding to neutral mucin. SDS-PAGE of purified protein (termed Mucus-Rs) under non reducing conditions showed it to be a homogenous glycoprotein (mol. wt. 250 kDa), while under reducing conditions, three bands corresponding to mol. wt. of 118,75 and 60 kDa were observed. Pretreatment of Mucus-Rs with pronase, trypsin and sodium metaperiodate markedly inhibited bacterial binding. GLC analysis of Mucus-Rs showed it to contain Mannose, Glucose, Galactose, Glucosamine, Galactosamine and Sialic acid as main sugars. Competitive binding in the presence of various sugars and lectins indicated the involvement of mannose in the mucus-bacterial interactions. The Mucus-Rs binding was highly specific for S. typhimurium; no significant binding was seen with E.coliand V. cholerae. Thus, we conclude that S. typhimurium specifically binds to a 250 kDa neutral mucin of intestinal tract. This binding appears to occur via specific adhesin-receptor interactions involving bacterial pili and mannose of neutral mucin.     

The biochemical composition, energy content, and chemical antifeedant defenses of the common Antarctic Peninsular sea stars Granaster nutrix and Neosmilaster georgianus

The sea stars Granaster nutrix and Neosmilaster georgianus are conspicuous members of benthic communities along the Antarctic Peninsula. An analysis of the proximate composition of somatic body components of nonreproductive adults indicates the nutrient storage organs (pyloric caeca) are rich in both protein (60.7 and 60.6% mean dry wt, respectively) and lipid (25.4 and 29.8% mean dry wt, respectively). Body-wall tissues, while containing small inconspicuous skeletal ossicles, are also comprised of significant levels of organic matter (33.5 and 55.7% mean dry wt, respectively), attributable primarily to protein. Both the pyloric caeca and body-wall tissues are relatively rich in energy (mean energy levels=24.8 and 26.5 kJ g⁻¹ dry wt; 8.4 and 14.1 kJ g⁻¹ dry wt, respectively). Despite the availability of these nutrients and energy neither sea star is preyed upon by the sympatric omnivorous sea star Odontaster validus, a common predator of other Antarctic sea stars. Laboratory feeding bioassays indicate that O. validus rejects live intact individuals and body-wall tissues of both sea star species while readily consuming dried krill. Alginate food pellets containing a krill powder and tissue level concentrations of organic methanol extracts of body-wall tissues were also rejected by O. validus. Moreover, the copious mucus released from the body wall of N. georgianus was deterrent in O. validus food pellet bioassays. Thus, both sea stars evidently possess defensive secondary metabolites that defend against predation and are likely to play a role in mediating materials and energy transfer in the Antarctic benthos.     

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel

The tritiated arylazido phenylalkylamine (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4, 5-trimethoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle transverse-tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a Kd of 2.0 +/- 0.5 nM and a Bmax of 17.0 +/- 0.9 pmol/mg protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high-affinity drug receptor carrying subunit of the skeletal muscle transverse-tubule L-type calcium channel.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

Biomass, litterfall and decomposition rates for the fringed rhizophora mangle forest lining the Bon Accord Lagoon, Tobago

The mangrove forest that fringes the Bon Accord Lagoon measures 0.8 km(2) and is dominated by red mangrove (Rhizophora mangle). This forest forms the landward boundary of the Buccoo Reef Marine Park in Southwest Tobago, and is part of a mangrove-seagrass-coral reef continuum. Biomass and productivity, as indicated by litterfall rates, were measured in seven 0.01 ha monospecific plots from February 1998 to February 1999, and decomposition rates were determined. Red mangrove above-ground biomass ranged between 2.0 and 25.9 kg (dry wt.) m(-2). Mean biomass was 14.1+/-8.1 kg (dry wt.) m(-2) yielding a standing crop of 11 318+/-6 488 t. Litterfall rate varied spatially and seasonally. It peaked from May to August (4.2-4.3 g dry wt. m(-2) d(-1)) and was lowest from October to December (2.3-2.8 g dry wt. m(-2) d(-1)). Mean annual litterfall rate was 3.4+/-0.9 g dry wt. m(-2) d(-1). Leaf degradation rates ranged from 0.3% loss d(-1) in the upper intertidal zone to 1% loss d(-1) at a lower intertidal site flooded by sewage effluent. Mean degradation rate was 0.4+/-1% loss d(-1) . The swamp produces 2.8 t dry wt. of litterfall and 12 kg dry wt. of decomposed leaf material daily. Biomass and litterfall rates in Bon Accord Lagoon were compared to five similar sites that also participate in the Caribbean Coastal Marine Productivity Programme (CARICOMP). The Bon Accord Lagoon mangrove swamp is a highly productive fringed-forest that contributes to the overall productivity of the mangrove-seagrass-reef complex.     

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.     

Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-l/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-l/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-l/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice, mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced express     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.     

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.     

Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules

A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.     

A multicopy suppressor of a cell cycle defect in S. pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein.

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIases) which have been implicated in intracellular protein folding, transport and assembly. Cyclophilins are also known as the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA). The most common type of cyclophilins are the 18 kDa cytosolic proteins containing only the highly conserved core domain for PPIase and CsA binding activities. The wis2+ gene of the fission yeast Schizosaccharomyces pombe was isolated as a multicopy suppressor of wee1-50 cdc25-22 win1-1, a triple mutant strain which exhibits a cell cycle defect phenotype. Sequence analysis of wis2+ reveals that it encodes a 40 kDa cyclophilin-like protein, homologous to the mammalian cyclophilin 40. The 18 kDa cyclophilin domain (CyP-18) of wis2 is followed by a C-terminal region of 188 amino acids. The C-terminal region of wis2 is essential for suppression of the triple mutant defect. Furthermore this region of the protein is able to confer suppression activity on the 18 kDa S.pombe cyclophilin, cyp1, since a hybrid protein consisting of an 18 kDa S.pombe cyclophilin (cyp1) fused to the C-terminus of wis2 shows suppression activity. We also demonstrate that the level of wis2+ mRNA increases 10- to 20-fold upon heat shock of S.pombe cells suggesting a role for wis2+ in the heat-shock response.     

Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase

A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.     

Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-1/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-1/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-1/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice. mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression of mfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistry assessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liver regeneration. These data suggested that MFREP-1 might play an important role in liver regeneration and be involved in the regulation of cell growth.