Radular myoglobin and protein variation within and among some littorinid species (Mollusca: Gastropoda)

The radular muscles of several littorinid species, including Littorina littorea, L. saxatilis, L. obtusata, L. striata and Melarhaphe neritoides, contain myoglobin (Mb). Here we report on the presence of radular Mb in eight other littorinids: L. compressa, L. arcana, L. fabalis, Nodilittorina punctata, N. trochoides, N. radiata, Littoraria undulata and Littoraria cingulifera. Using native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) we compared the Mb and soluble protein (SP) profiles of these species. This suggested that: (1) L. saxatilis and L. arcana may have specific Mb/SP profiles, (2) Littoraria spp., Nodilittorina spp. and L. striata share similar Mb patterns, (3) Mb is remarkably diverse in the genus Littorina, (4) L. littorea shows intraspecific Mb/SP variation, (5) L. saxatilis does not show geographic Mb/SP differences, and (6) IEF uncovers substantial hidden Mb/SP heterogeneity not shown by PAGE (particularly for Melarhaphe neritoides). Hence, littorinid Mb/SP may be a useful taxonomic marker whose ecophysiological significance deserves further study, even if its genetic basis remains unclear.     

Synthesis of Aspartyl Tripeptide Esters in Relation to Structural Features of Sweet Peptides

The distribution of multiple forms of β-amylase in some varieties or species of soybean seeds was examined by the gel isoelectric focusing method. Seven components (1′, 1, 2, 3, 4, 5 and 6) were found. Their respective isoelectric points were 5.07, 5.15, 5.25, 5.40, 5.55, 5.70 and 5.93±0.04. The varieties or species of soybean seeds were separated into two types by their zymograph: the low pI type and high pI type. Component 6 was purified from commercial defatted soybean meal containing all seven components by ion-exchange column chromatography and by gel filtration, and compared with previously purified components 2 and 4. Components 2, 4 and 6 had the same molecular weight and immunological properties but some differences were found in their amino acid compositions and enzymatic properties. The C-terminal amino acid of components 2 and 6 was glycine but that of component 4 was alanine. It was concluded from these results that differences between components 2, 4 and 6 were caused by charged amino acid substitution.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

The isolation of carcinogen-binding protein from livers of rats given 4-dimethylaminoazobenzene

1. Three azo-dye-binding proteins were identified in the soluble cell supernatant fraction from livers of rats that had received 4-dimethylaminoazobenzene by intraperitoneal injection. 2. One is basic and was highly purified. It has an isoelectric point of pH8.4 in barbital-sodium chloride buffer, I0.1, an S(20,w) value of 3.5s and a molecular weight determined by Sephadex chromatography of 45000. 3. It does not have N-terminal amino acids with free alpha-amino groups. 4. Digestion with Pronase gives rise to a single azo-dye-bound peptide, which on hydrolysis is shown to contain glycine, alanine, serine, threonine, glutamic acid and aspartic acid. The amino acid that binds the azo-dye was not identified. 5. On starch-gel electrophoresis the basic protein separates into a double band, indicating microheterogeneity. 6. The other two proteins were partially purified and occur in a fraction together. They have isoelectric points near neutrality and a molecular weight as determined by Sephadex chromatography of 13800. 7. The absorption spectra in formic acid of both the basic and the low-molecular-weight proteins are similar. The azonium ion has an absorption maximum at 518mmu and another adsorbed chromogen is present with an absorption maximum at 395mmu.     

Studies on myoglobin from the finback whale (Balaenoptera physalus). Preparation, physicochemical and immunochemical characterization, differentiation from sperm-whale myoglobin, amino acid composition and end-terminal analyses

1. Crystalline myoglobin was isolated from the skeletal muscle of the finback whale and fractionated, in its cyanmet form, into nine components (I-IX) by chromatography on CM-cellulose. Also in the cyanmet form, it was resolved into six components by electrophoresis on starch gel. Correspondence between the electrophoretic and chromatographic components was determined, and interconversion between components revealed by chromatography and electrophoresis. 2. The chromatographic myoglobin components were homogeneous in the ultra-centrifuge. Molecular weights of certain components were determined by means of sedimentation equilibrium and by gel filtration on Sephadex G-100. Values from these two methods corresponded to the minimum molecular weight calculated from the iron content. 3. The spectral properties of the chromatographic components were investigated in the visible and the ultraviolet ranges. 4. The major components of finback-whale myoglobin and sperm-whale myoglobin showed almost identical spectral, electrophoretic and chromatographic behaviours, but had different infrared spectra. The infrared spectra of the corresponding apoproteins were almost identical. 5. Rabbit antisera to sperm-whale myoglobin component X cross-reacted with finback-whale myoglobin components V, VI and VII only about 30%. 6. The major chromatographic components of finback-whale myoglobin have identical amino acid compositions. The polypeptide chain contains 151 amino acid residues and its molecular weight is 17504. 7. The N-terminal end of the chain is: [Formula: see text] Amino acids released from myoglobin by the action of carboxypeptidase A at different intervals were determined.     

Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin

The colour of buffalo (Bubalus bubalis L.) meat is darker than bovine meat. Since meat colour depends on the concentration of myoglobin (Mb) and its oxidation state, we have determined the main structural and functional properties of buffalo Mb. Buffalo Mb was purified from longissimus dorsi muscles and its molecular mass determined by ESI Q-TOF mass spectrometry. The molecular mass 17,034.50 was 86.20 Da higher than the bovine Mb. This was confirmed by analysing its primary structure, using a combined approach based on Edman degradation and MALDI-TOF mass spectrometry. Comparing the amino acid sequences of both Mbs, we found three amino acid differences out of 153 amino acid residues. One is a conservative substitution (D(bov)141E(buf)), and the other two (A(bov)19T(buf) and A(bov)117D(buf)) are nonconservative. These amino acid substitutions are unlikely to cause structural changes because they are located far from the heme binding pocket, as revealed by the 3D structure of buffalo Mb elaborated by homology modelling. Stability analyses show no difference with the bovine Mb for helix E and only minor differences in the stability values for helices A and G. Moreover, autoxidation rates of purified buffalo and bovine myoglobins at 37 degrees C, pH 7.2, were almost identical, 0.052+/-0.001 h(-1) and 0.054+/-0.002 h(-1), respectively, as were their oxygen-binding Kd values, 3.7+/-0.1 microM and 3.5+/-0.1 microM, respectively. The percent of MetMb values were almost identical. The results presented here suggest that the darker buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393+/-0.005 g/100 g of tissue) and consequently MetMb, which are almost twice as high as bovine meat (Mb: 0.209+/-0.003 g/100 g of tissue).     

Lack of significant esterase and myoglobin differentiation in the periwinkle,Littorina striata (Gastropoda,Prosobranchia)

The relationship between gene flow and the maintenance of geographic or morphology-related variation in the polymorphic Macaronesian periwinkle, Littorina striata, was investigated by means of isoelectric focusing of esterases (EST) and myoglobin (Mb). This revealed that: (1) individual EST variation is very high, (2) there is no EST differentiation between sexes, shell morphotypes or wave-exposure regimes, (3) there is no clear macrogeographic patterning of EST variability, although both Cape Verde Islands have the highest, with exception of Flores, mean number of EST bands, and (4) there is no Mb variation, not even between islands separated by more than 2000 km. These results indicate that L. striata shows a high degree of genetic homogeneity among geographic populations and that the morphological patterning in this species persists in the presence of intense gene flow.     

The isolation, characterization and amino terminal sequence of the vitamin D-binding protein (group specific component) from mouse plasma

1. In order to establish a homologous system in which to study the interaction of mouse vitamin D-binding protein (MVDBP) with mouse T-cell lymphocytes, we purified MVDBP from mouse plasma. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified MVDBP had an apparent relative molecular weight of 49,000. 3. Previous work in our laboratory has shown that purified rat vitamin D-binding protein (RVDBP) has an apparent relative molecular weight of 52,000. 4. The amino terminal amino acid sequence of MVDBP is shown below and compared with that of RVDBP. MVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysAsnGluLeuAlaMetLeuGlyLysGlu RVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLysAsp AspPhe AspPhe While 21 out of 24 residues (87.5%) of the amino terminus of MVDBP are the same as those in RVDBP, residues 14, 17, 18 and 22 (underlined) are different. 5. The sedimentation coefficient of the protein, determined by sucrose density gradient ultracentrifugation, is 3.8 for MVDBP and 4.1 for the rat VDBP. 6. The MVDBP purified in this study exhibits only one isoform on isoelectric focusing; the isoelectric point was 4.87 as determined on pH 4.0-6.5 isoelectric focusing gels (IEF). 7. The binding of vitamin D3, 25-hydroxyvitamin D3 and three other analogs was investigated with a charcoal dextran assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

H2O2-mediated cross-linking between lactoperoxidase and myoglobin: elucidation of protein-protein radical transfer reactions

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.     

Exploring the molecular basis of heme coordination in human neuroglobin

Neuroglobin (Ngb), a recently discovered ancient heme protein, presents the typical globin fold and is around 20% identical to myoglobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

Properties of common wheat ferredoxin, and a comparison with ferredoxins from related species of triticum and aegilops.

Wheat ferredoxin was purified from the leaves of common wheat (Triticum aestivum). The absorption spectrum showed maxima at 465, 425, 332, and 278 nm. The absorbance ratio, A425 nm/A278 nm was 0.49, and the millimolar extinction coefficient at 425 nm was 10.8 mM-1. cm-1. The amino acid composition was determined to be Lys5, His2, Arg1, Asp11, Thr5, Ser7, Glu18, Pro5, Gly6, Ala7, Cys5, Val7, Met1, Ile4, Leu7, Tyr4, Phe1, and Trp1. The total number of amino acid residues was 97. The molecular weight was calculated from the amino acid composition to be 10,829, including iron and sulfur atoms. This value was confirmed by other methods, which were based on the contents of non-heme iron and of terminal amino acid. The N-terminal amino acid was alanine, and the C-terminal amino acid sequence was -Glu-Leu-Thr-AlaCOOH. Comparative studies were performed between T. aestivum ferredoxin and ferredoxins isolated from closely related species; these were T. aegilopoides, T. durum, Ae. squarrosa, and Ae. ovata. No significant differences in the properties of these ferredoxins were detected. It was also shown that these ferredoxins are immunologically homologous. It is, therefore, likely that one molecular species of ferredoxin is distributed through two genera of Triticum and Aegilops.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 145–151 (antigenic site 5) of myoglobin

Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145–151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs—bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)—have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145–151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145–151 mAbs are due to the effects of amino acid substitutions outside the region 145–151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145–151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15–22, 56–62, 94–100, and 113–120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected.     

Isolation and characterization of major intrinsic microsomal membrane proteins.

Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.     

Purification and characterization of rhizopuspepsin isozymes from a liquid culture of Rhizopus chinensis

1. Rhizopuspepsin has been purified from liquid cultures of Rhizopus chinensis. 2. Purification by ammonium sulfate precipitation, affinity chromatography on pepstatin Sepharose and low/high resolution isoelectric focusing produced five isoelectric forms. 3. The two major isozymes pI 5.1 and 5.8 did not differ significantly in amino acid composition, molecular weight and enzyme activity. 4. Three minor isozymes were partially purified as pI 7.35, 7.41 and 7.9.     

A comparison of the physical and chemical properties of four cytochromes c from Azotobacter vinelandii

1. A modified method for the separation and purification of four cytochromes c from Azotobacter vinelandii is described. Two new cytochromes c have been purified and are designated cytochromes c(551) and c(555). 2. Additional evidence is presented to establish the dihaem nature of cytochrome c(4). Ultracentrifugation data indicated similar molecular weights for the native and the denatured protein. Cleavage with CNBr yielded seven peptides; the amino acid compositions of the purified peptides were determined. Only one haem peptide was recovered. 3. Cytochromes c(551) and c(555) were characterized as acidic proteins of molecular weights about 12000. The spectral properties, isoelectric points, ;maps' of peptides from CNBr cleavage and amino acid compositions were determined for these two proteins. 4. The spectral properties, isoelectric points, molecular weights, CNBr peptide ;maps', amino acid compositions, relative oxidation-reduction potentials and e.p.r. (electron-paramagnetic-resonance) spectra of the four cytochromes c were compared. Cytochrome c(4) and cytochrome c(551) appear to be distinct proteins. The distinction between cytochromes c(5) and c(555) was not as clear, and our data are inadequate to establish firmly that they are distinct proteins. 5. The dihaem nature of cytochrome c(4) is evident in its e.p.r. spectrum. The e.p.r. spectra are similar to the spectra of mammalian cytochromes c.     

Mechanisms of reoxygenation injury in myocardial infarction: implications of a myoglobin redox cycle

The addition of ascorbate to ischemic rat hearts prevents the myocardial damage associated with reoxygenation. H2O2 oxidizes myoglobin (Mb+2) to higher oxidation states (Mb+4 and Mb+5) which are rapidly reduced by ascorbate. It is proposed that the operation of a myoglobin redox cycle, in which H2O2 causes the two-electron oxidation of myoglobin, is a critical determinant of reperfusion injury. Conversely, the reduction of myoglobin, in one-electron steps, may represent an essential protective mechanism against such injury in the heart.     

Palmitate interaction with physiological states of myoglobin

Background

Previous studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III) MbCN. The present study has observed PA interaction with physiological states of Fe(II) Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport.

Methods

¹H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding.

Results

Palmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15 ppm upfield shift of the PA methylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant, which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein (FABP).

Conclusions

Given contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein (FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid.

General significance

Under oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism.     

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.