Determination of the complete cDNA sequence, construction of expression systems, and elucidation of fibrinolytic activity for Tapes japonica lysozyme

The lysozyme of the marine bivalve, Tapes japonica (13.8 kDa), belongs to the invertebrate lysozyme family and displays both chitinase and isopeptidase activities. We determined the complete cDNA sequence and constructed effective expression systems for this enzyme using Escherichia coli (BL21) and Pichia pastoris. The native and recombinant proteins indicated lysozyme activity and isopeptidase activity, including the proteolysis of d-dimer, a plasminolytic product of stabilized polymeric fibrin. These results will be utilized for the structural and functional study of invertebrate lysozymes, and for the development of applications for thrombosis therapies.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii.

Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and cyclic adenosine 3',5'-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks(I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle. Peak III utilizes histone as substrate and is activated by cyclic AMP.     

Resolution of myocardial phospholipase C into several forms with distinct properties.

Phospholipase C activity capable of hydrolysing phosphatidylinositol in bovine heart was resolved into four forms (I-IV) by ion-exchange chromatography. Some of these forms could only be detected if the assay was performed at acidic pH (I and IV) or in the presence of deoxycholate (II). Gel-filtration chromatography indicated that the four forms had different molecular weights in the range 40000-120000. I, II and III all had pH optima in the range 4.5-5.5. However, the major form (III) also had substantial activity at pH 7.0 and above. The activities of I, II and III at pH 7.0 were stimulated by deoxycholate; this effect was most marked with I and II, which had very low activity at this pH. All forms of the enzyme were inhibited by EGTA and required 2-5 mM-CaCl2 for maximal activity. When the fractions eluted from the ion-exchange and gel-filtration columns were assayed with polyphosphoinositides as substrates there was a close correspondence to the elution profile obtained with phosphatidylinositol as substrate; there was no evidence for the existence in heart of phospholipase C activities specific for individual phosphoinositides.     

Testicular protein kinases. Characterization of multiple forms and ontogeny.

Protein phosphokinase activity from the cytosol (105,000 X g soluble fraction) of testes from sexually mature rats has been resolved be DEAE-cellulose chromatography in three forms of protein kinase, cAMP-dependent protein kinases I and II and cAMP-independent protein kinase III. Adenosine 3':5'-monophosphate-binding activity (cAMP-binding activity) was associated with protein kinases I and II but not with protein kinase III. Protein kinases I, II, and III exhibited different pH optima, cyclic nucleotide dependency, and relative substrate specificity. Protein kinases I and II were inhibited by a heat-stable protein inhibitor from rat skeletal muscle, whereas protein kinase III was not inhibited. According to previously established criteria (Traugh, J. A., Ashby, C.D., and Walsh D. A. (1974) Methods Enzymol. 38, 290-299) protein kinases I and II can be classified as cAMP-dependent holoenzymes consisting of regulatory and catalytic subunits. Protein kinase III is a cAMP-independent protein kinase.     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C-terminal deletions

To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37 degrees C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3' end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57-42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (mlDL): Structure-function relationship

Based on the three-dimensional model of the bifunctional enzyme destabilase-lysozyme of the medicinal leech (mlDL) in complex with trimer of N-acetylglucosamine (NAG)₃ by site-directed mutagenesis method, the functional role of the group of amino acids (Glu14, Asp26, Ser29, Ser31, Lys38, His92) in manifestation of lysozyme (glycosidase, muramidase) and isopeptidase activities has been investigated by site-directed mutagenesis. The results obtained go well with hypothesis, that lysozyme active site of mlDL includes catalytic Glu14 and Asp26 residues, and isopeptidase site functions as Ser/Lys catalytic dyad presented by catalytic residues Ser29 and Lys38. Thus, among the invertebrate lysozymes, mlDL presents the first example of a bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

Effective method for purification of lysozyme from human urine

Lysozyme in the urine of a hemodialysis patient was purified in two steps: DEAE Sephadex chromatography followed by Sephacryl chromatography. The Sephacryl S-100 column chromatographed fraction showing lytic activity was proven to give one band on SDS-PAGE and to have a molecular mass of 14 500, in agreement with that of lysozyme. The N-terminal amino acid sequence of this purified protein was identical to that of lysozyme. These results indicate that the protein purified was indeed lysozyme. The specific affinity of lysozyme for Sephacryl S-100 may explain the greater purity of the same protein isolated by this method.     

Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation

Interactions of types I, II, and III protein kinase C (PKC) with phospholipids were investigated by following the changes in protein kinase activity and phorbol ester binding. The acidic phospholipids such as phosphatidylserine (PS), phosphatidic acid, phosphatidyl-glycerol, and cardiolipin, which are activators of PKC in the assay of protein phosphorylation, could differentially inactivate PKC I, II, and III during preincubation in the absence of divalent cation. The phospholipid-induced inactivation of PKC was concentration and time dependent and only affected the kinase activity without influencing phorbol ester binding. PKC I was the most susceptible to the phospholipid-induced inactivation, and PKC III was the least. The IC50 values of PS for PKC I, II, and III were 5, 45, and greater than 120 microM, respectively. Addition of divalent cation such as Ca2+ or Mg2+ suppressed the phospholipid-induced inactivation of PKC. In the absence of divalent cation, PKC I, II, and III all formed complexes with PS vesicles, although to a slightly different degree, as analyzed by molecule sieve chromatography. [3H]Phorbol 12,13-dibutyrate binding for PKC I, II, and III was recovered after chromatography; however, the kinase activities of all these enzymes were greatly reduced. In the presence of Ca2+, all three PKCs formed complexes with PS vesicles, and both the kinase and phorbol ester-binding activities of PKC II and III were recovered following chromatography. Under the same conditions, the phorbol ester-binding activity of PKC I was also recovered, but the kinase activity was not. The phospholipid-induced inactivation of PKC apparently results from a direct interaction of phospholipid with the catalytic domain of PKC; this interaction can be suppressed by divalent cations. In the presence of divalent cations, PS interacted preferentially with the regulatory domain of PKC and resulted in the activation of the kinase.     

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.     

Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11 degrees and 25 degrees C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS-PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. Copyright 1999 Academic Press.     

Epidermal growth factor stimulates protein kinase activity, specific for ribosomal protein S6 in loach embryo blastoderm

The activity of a protein kinase specific to ribosomal protein S6 was determined in early loach embryos in basal conditions and after their treatment with epidermal growth factor (EGF). The cytosol of loach blastoderms isolated at the early gastrula stage possessed a high level of protein kinase activity catalysing incorporation of 0. 33 pmol.min-1.mg-1 of Pi into exogenous S6 protein of rat liver ribosomal 40S subunit. The treatment of embryos for 30 min with EGF (10 ng/ml) added to the incubation medium caused an 25% increase of total S6-kinase activity in cytosol compared with its counterpart in non-stimulated embryos. After chromatography of loach embryos cytosol on DE-52 three fractions possessing S6-kinase activity were revealed, which were eluted with 10 microM cAMP (I), 150 mM NaCl (II) and 300 mM NaCl (III), respectively. After treatment of blastoderms with EGF in the described conditions the enzymatic activity 2-fold decreased in fraction I, increased in fraction II 4-fold and remained practically unchanged in fraction III. The mitogen-stimulated kinase, apart from S6 protein, phosphorylated also casein and but not histone H1.     

Studies of dynein from Tetrahymena cilia using agarose polyacrylamide gel electrophoresis.

Described in this report is an application of agarose-polyacrylamide gel electrophoresis, which separates protein components of crude dynein fraction (Fraction I by Gibbons) derived from Tetrahymena cilia. By this method, the fraction was separated into three protein components (designated as bands I, II and III) on the gel. When the gel was actively stained for dynein ATPase, a single band appeared, which coincided with the position of band I. A purified dynein prepared by controlled pore glass (CPG-10) column chromatography and followed by Biogel A-15m filtration showed one band on the gel at the same position as band I. These results suggest that among these three protein components, band I represents dynein and bands II and III are derived form non-ATPase protein. 'Burstic phenomenon' was also observed on their ATPase activity when axoneme or crude dynein fractions were used for ATPase assay, while the phenomenon was almost extinguished when partially purified dynein after controlled pore glass column chromatography was used as sample.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Structure of Bordetella pertussis peptidoglycan.

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing [3H]diaminopimelic acid and treated by a hot (96 degrees C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and less than 2% protein. Radiochemical analyses indicated that 3H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of greater than 95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl++ +-alanine.     

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS)

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.