Non-dissociating Conditions Research Articles

Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica.

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.

Polyacrylamide gel electrophoresis of soybean seed proteins

Four major and 14 minor protein bands were detected when total salt soluble proteins of soybean (Glycine max cultivar Warszawska) seed were subjected to polyacrylamide gel electrophoresis under nondissociating conditions, and 16 protein bands were detected under dissociating conditions. Molecular weights of three major protein fractions in PAGE SDS were determined for around 18 500, 36 000 and 80 000 daltons.

Insulin-degrading enzyme hydrolyzes ATP.

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.

Chaperoning of Insertion of Membrane Proteins into Lipid Bilayers by Hemifluorinated Surfactants: Application to Diphtheria Toxin

Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid vesicles. To test the ability of HF-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of the diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used Förster resonance energy transfer to follow the effect of HF-TAC on aggregation of aqueous and membrane-bound T-domain. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of the aqueous T-domain at low pH. The presence of HF-TAC in the buffer is demonstrated to suppress aggregation in solution and ensure correct insertion and folding of the T-domain into the lipid vesicles, without solubilizing the latter. Thus, hemifluorinated surfactants stabilize the low-pH conformation of the T-domain as a water-soluble monomer while acting as low-molecular weight chaperones for its insertion into preformed lipid bilayers.

Synthesis and Preliminary Biochemical Assessment of Ethyl‐Terminated Perfluoroalkylamine Oxide Surfactants.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and preliminary biochemical assessment of ethyl-terminated perfluoroalkylamine oxide surfactants

The synthesis and usefulness in membrane biochemistry of a new class of surfactants have been investigated. 1-Ethyl-2-dimethylamine oxide polar heads were grafted onto a hydrocarbon, a fluorocarbon or an ethyl-capped fluorocarbon hydrophobic tail. The ability of the resulting surfactants to extract and/or to stabilize in aqueous solution a test membrane protein, cytochrome b 6 f, was evaluated. While it is not a detergent, the hemifluorinated derivative efficiently kept purified cytochrome b 6 f soluble, native and functional. The data suggest that alkyl-capped fluorocarbon surfactants provide an interesting alternative to classical detergents for handling membrane proteins in aqueous solutions under non-dissociating conditions.

ラット心筋の局所的ミオシンアイソザイム構成比に及ぼすランニングトレーニングの影響

We examined the effect of running training on regional cardiac myosin isozyme composition in rats. Male Sprague-Dawley strain rats (4 weeks old) were used, and divided into two groups: sedentary control (C) and trained (T) groups. The T group was trained by treadmill running (40 m/min, 1h/day, 5 days/week, for 12 weeks) . At 16 weeks old, their hearts were excised. The left ventricle was separated into the subendocardium (Endo) and subepicardium (Epi) by dissecting the ventricle at the mid-wall. The ventricular myosin isozymes were examined by electrophoresis on pyrophosphate gel under non-dissociating conditions. The results showed the following: 1) The relative heart weight of the T group was significantly higher than the C group. 2) Left ventricular myosin isozyme composition showed a region-specific distribution in the C rats, and the proportion of V3 myosin or β-myosin heavy chain in the Endo was significantly higher than that of the Epi. However, the training had no effect on the cardiac myosin isozyme in either portion. 3) The activity of citrate synthase did not show transmural gradient in the ventricle of C animals. Training had no effect on the activities of either portion. 4) The activity of lactate dehydrogenase (LDH) showed transmural gradient in the ventricle of C rats. Training-induced changes in the activity of LDH were found in both portions, therefore, training abolished the transmural gradient in the activity of LDH, suggesting a corresponding redistribution of the myocardial work load.These results indicate that running training might induce the redistribution of the myocardial work load, whereas the stimulation apparently has no effect on the regional distribution of cardiac myosin isozyme composition.

Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important information about the composition of porcine skeletal muscle. The combination of the two methods may be essential to accurately characterize porcine skeletal muscles.

Isolation and Characterization of a New Member of the Scavenger Receptor Superfamily, Glycoprotein-340 (gp-340), as a Lung Surfactant Protein-D Binding Molecule

We have purified a previously unknown glycoprotein (designated gp-340) from human bronchioalveolar lung washings from a patient with alveolar proteinosis. gp-340 was identified by its calcium-dependent binding to lung surfactant protein D (SP-D) and by its molecular mass of 340 kDa in the reduced state on SDS-polyacrylamide gel electrophoresis (PAGE). gp-340 was purified from the 10,000 x g pellet of the lavage fluid by ion-exchange and gel permeation chromatography. On SDS-PAGE, gp-340 showed an apparent molecular mass of 290 kDa in the unreduced state. On gel chromatography under non-dissociating conditions, the apparent molecular mass of gp-340 was >1000 kDa. The presence of N-linked glycosylation was shown by digestion with N-glycosidase F, which reduced the apparent molecular mass of gp-340 under reducing condition to about 300 kDa. Partial amino acid sequence data showed the presence of scavenger-receptor type domains. Monoclonal and polyclonal antibodies were raised against gp-340, and their specificities were confirmed by Western blotting. The antibodies were used for immunohistochemical localization of gp-340 in the lung, where it was found on the surface of and within alveolar macrophages. Direct binding between gp-340 and SP-D took place at physiological ionic strength, required the presence of calcium, and was not inhibited by maltose. The binding between SP-D and mannan also required the presence of calcium, but this interaction was completely inhibited by maltose. The same binding pattern was seen between gp-340 and recombinant human SP-D composed of the trimeric neck region and three carbohydrate recognition domains. These findings indicate that the binding between gp-340 and SP-D is a protein-protein interaction rather than a lectin-carbohydrate interaction and that the binding to gp-340 takes place via the carbohydrate recognition domain of SP-D. We conclude that gp-340 is a new member of the scavenger-receptor superfamily and likely to be a truncated form of a receptor for SP-D.

Purification and binding properties of a human ficolin-like protein

Ficolin was initially identified from porcine uterus as a TGF- β1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca 2+. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification.

Changes in myosin heavy chain (MHC) expression during pregnancy in rat uterus.

Expression of myosin heavy chain (MHC) was investigated in rat uterus during pregnancy. Two MHC isoforms, SM MHCI (204 kDa) and SM MHCII (200 kDa), were resolved following the analysis of Guba Straub extract of non-pregnant and pregnant uterus using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Whereas SM MHCI showed an increase from 69.26 +/- 3.26% in nonpregnant uterus to 81.40 +/- 5.36% in pregnant uterus, the SM MHCII exhibited a corresponding decline from 30.73 +/- 3.29 to 18.59 +/- 5.36%. The native myosin separated under non-dissociating conditions and subsequently analyzed in denaturing SDS-PAGE also demonstrated two MHC isoforms with identical electrophoretic mobilities. A SDS-PAGE analysis of native myosin from pregnant rats extracted at room temperature and in the absence of proteolytic inhibitors revealed a characteristically increased proteolysis of MHC into two peptide products of 153 and 140 kDa during pregnancy. Such a proteolysis of MHC, but in very low proportions, was noticed in nonpregnant uterus too.

Purification and characterization of α-glucosidase complex from the intestine of the frog, Rana japonica

The enzymes responsible for much of the isomaltase and maltase activities in the intestine of the frog, Rana japonica, were purified by detergent solubilization and affinity chromatography on Sephadex G-200 gel. The two activities paralleled each other during purification. The isomaltase, maltase and glucoamylase activities eluted in the same pattern on Sepharose 4B gel filtration as well as on Sephadex G-200 gel affinity chromatography. Anti-rabbit sucrase-isomaltase antibody inhibited the isomaltase activity but not the maltase or glucoamylase activity of the purified enzyme preparation, while the three activities were precipitated in parallel by the antibody. The isomaltase activity was more stable at 55°C than the maltase and glucoamylase activities. On SDS-polyacrylamide gel electrophoresis under nondissociating conditions the purified enzyme preparation showed only one major band of 330 kDa, while under dissociating conditions it showed two bands of 116 and 212 kDa. These results suggest that isomaltase (apparently with no or minor maltase activity) is due to a protein domain (or protein) different from one which is responsible for maltase and glucoamylase activities. This implies that isomaltase is associated with maltase/glucoamylase to form α-glucosidase complex in the brush border membrane of the frog intestine.

Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein.

It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.

A recombinant polypeptide, composed of the α-helical neck region and the carbohydrate recogniton domain of conglutinin, self-associates to give a functionally intact homotrimer

A recombinant polypeptide composed of the α-helical neck region and carbohydrate recognition domain (CRD) of bovine conglutinin was expressed in Escherichia coli. The recombinant protein formed inclusion bodies but could be solubilised using a denaturation-renaturation cycle based on urea and then purified by affinity chromatography on a TSK- N-acetylglucosamine column. The purified product behaved as a homotrimer in nondissociating conditions, with three CRDs held together by the α-helical neck regions. The trimer, although lacking the N-terminal and collagen regions of the native conglutinin, showed the same binding carbohydrate specificities as the native molecule, for the complement fragment C3b and for lipopolysaccharides derived from Gram-negative bacteria.

Properties of Troponin C Acetylated at Lysine Residues

We have studied the properties of rabbit skeletal troponin C (TnC) fully acetylated at its lysine residues (AcTnC). Acetylation causes a decrease in thermal stability of both domains of TnC in the absence of Ca2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal domain monitored by far-UV circular dichroism is decreased by 16.3 degrees C. In the presence of 1 mM CaCl2, no cooperative unfolding can be detected up to 90 degrees C for either TnC or AcTnC. At 25 degrees C, CD spectra show that AcTnC has a slightly lower alpha-helix content in the absence of Ca2+, but higher in the presence of Ca2+ as compared to unmodified TnC. Acetylation causes a 3.5-fold increase in affinity for Ca2+ at the low-affinity sites and a 2-fold decrease at the high-affinity sites. Polyacrylamide gel electrophoresis under nondissociating conditions (no SDS, no urea, pH 8.6) indicates that acetylation has little effect on the apparent affinity of TnC for troponin I; however, the binding of the acetylated peptides corresponding to the N-terminal domain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enhanced, the altered properties of the N-terminal domain being predominantly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetylation increases TnC's affinity for myofibrils in the presence of Ca2+ approximately 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of myofibrils containing AcTnC is shifted to lower Ca2+ concentrations, consistent with the higher Ca2+ affinity of AcTnC at the low-affinity sites. These data indicate that positively charged lysine side chains, especially those located in the N-terminal domain, modulate TnC's structural stability and interactions with Ca2+ and other troponin components.

Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q.

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD. The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and some properties of soluble β-fructofuranosidase from larval midgut of the silkworm, Bombyx mori

Soluble sucrase was purified from midgut of the silkworm, Bombyx mori, to near homogeneity by ammonium sulphate fractionation at 40–80% saturation, column chromatographies of ion-exchange cellulose, hydroxylapatite and Sephacryl S-100 HR. Specific activity of 384.7 U/mg protein of the purified soluble sucrase was the highest activity among the sucrases so far purified from insects. The purified soluble sucrase hydrolysed sucrose, raffinose and stachyose, but never hydrolysed any other di- and trisaccharides, and derivatives of glucoside. The results indicate that the purified soluble sucrase is a β-fructofuranosidase. Molecular weight under non-dissociating conditions of the soluble β-fructofuranosidase, was estimated to be 85,000, and the subunit molecular weight by SDS-PAGE was 58,000. It appears that the soluble β-fructofuranosidase is a homodimer. The pH optimum of the soluble β-fructofuranosidase was pH 6.2 and 6.0–7.0, when sucrose and raffinose were used as substrates, respectively. The K m values of the soluble β-fructofuranosidase were 6.5 and 8.6 mM, when sucrose and raffinose were used as substrates, respectively. The effect of metal ions on the soluble β-fructofuranosidase was inhibitory in 13 metal ions investigated. The notable ones were Hg 2+, Cu 2+, Pb 2+, Fe 3+ and Cd 2+, affecting activity to reduce 0–20% of the control activity. Tris and tricine were potent inhibitors for the soluble β-fructofuranosidase and inhibitor constant ( k i) of Tris was 9.2 mM when sucrose was used as a substrate. After enzymatic reaction of the soluble β-fructofuranosidase with sucrose as a substrate, a compound corresponding in mobility to trisaccharides such as raffinose and melezitose was identified in the reaction mixture, which indicated a transferolytic activity of the soluble β-fructofuranosidase. Stability of the soluble β-fructofuranosidase against various pHs preserved for 48 hr at 37°C was investigated. Ninety percent, and more than 50% of the initial activity, was maintained at pH 6.5 and in the range pH 4.5–9.5, respectively, but almost no activity was detected at pH 4.0.

Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein

A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose >> GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP.

Purification and characterization of bovine mannan-binding protein.

Bovine mannan-binding protein (bMBP) was observed in serum by its Ca(2+)-dependent binding to mannan and by an M(r) of 28 kDa under reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by precipitation with polyethyleneglycol (PEG), affinity chromatography on mannan-Sepharose eluted with EDTA, and absorption on Sepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies. Fractions containing the lectin were reapplied to mannan-Sepharose and eluted first with N-acetyl-D-glucosamine (GlcNAc) to remove conglutinin, and then with mannose to elute the 28 kDa lectin. Further purification was achieved by ion-exchange chromatography on Mono-Q and by mannose-gradient elution from a mannan-Sepharose column. SDS-PAGE of the purified lectin showed three high molecular weight bands under non-reducing conditions. The reduced protein gave a single band of 28 kDa. On gel permeation chromatography under non-dissociating conditions, the protein emerged at a volume corresponding to M(r) approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, and a high glycine content (17.7%), suggesting the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The N-terminal 26 amino acids showed 62% identity with human MBP, when three gaps were allowed in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Different biventricular remodelling of myosin and collagen in pulmonary hypertension.

1. To clarify the metabolism of contractile and non-contractile proteins of the ventricles during the development of right ventricular hypertrophy (RVH) and accompanying congestive heart failure (CHF) in response to a pressure overload, monocrotaline was injected subcutaneously into Sprague-Dawley (SD) rats. Myosin isoenzymes (MIE) were analysed by pyrophosphate gel electrophoresis under non-dissociating conditions. Acid-soluble collagens were analysed by an improved, noninterrupted sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tissue collagen content was also measured after the estimation of hydroxyproline concentration in tissues. 2. Monocrotaline induced RVH, but not left ventricular hypertrophy, at 2 weeks after the injection of monocrotaline, with severe RVH with CHF present at 4 weeks. In the right ventricles (RV) treated with monocrotaline, MIE shifted significantly from V1 to V3 at 2 weeks. The shift of MIE was more pronounced at 4 weeks. The proportion of type III collagen increased significantly compared with controls at 2 weeks. At 4 weeks, the proportion of types III and V collagens increased significantly compared with controls. 3. In left ventricles (LV) treated with monocrotaline, a similar but less remarkable shift of MIE was observed without remodelling of collagen types at 2 and 4 weeks. The concentration of collagen in either the RV or LV treated with monocrotaline showed no significant changes at 2 and 4 weeks compared with controls. 4. These results demonstrate a remodelling of the contractile and non-contractile proteins during the development of RVH and accompanying CHF, and provide evidence for changes in protein metabolism of the counterpart of RV (i.e. the LV). These results may provide important insights into the pathophysiology of adaptive or non-adaptive cardiac hypertrophy in response to a pressure overload.