Cloning, expression and characterization of horse L-ferritin in Escherichia coli.

Horse L-ferritin cDNA was cloned from horse liver, and the base sequence was determined. The L-ferritin was expressed using pTZ18U encoding lac promoter, and found to possess an additional 8-amino acid sequence at the N-terminus as compared with commercially obtained horse spleen (natural) ferritin. It was determined that there was Pro at position 94 in both the recombinant and natural L-ferritin, although it was previously reported that Leu was in this position in the natural species. Transmission electron microscopy showed that this recombinant ferritin formed a 24-mer shell.

Heterogeneities in ferritin dimers as characterized by gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques.

To understand the mechanism underlying the preferential dimerization of ferritin shells, we studied monomers and dimers from both horse spleen and recombinant horse L-apoferritin by using gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques. Our study of the kinetics of dimer-monomer dissociation that is produced by heating revealed the presence of at least two types of dimers, namely, weakly and strongly linked dimers with activation energies of 124 +/- 14 and 157 +/- 16 kJ/mol, respectively. Our study using thiol reagents indicated that the dimerization in horse spleen ferritin is partially mediated by disulfide bridges being formed between H-chains. Our analysis of the components that resulted from the dimer-monomer dissociation further clarified that these dimers form interdigitation structures. In summary, five types of dimers were identified in horse spleen apoferritin: reversible dimers with very weak interaction, non-sulfide dimers with weak interaction, non-sulfide dimers with strong interaction, disulfide dimers linked only by disulfide bridges, and disulfide dimers linked by disulfide bridges and having other interactions.

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Amide proton titration shifts in bull seminal inhibitor IIA by two-dimensional correlated 1H nuclear magnetic resonance (COSY). Manifestation of conformational equilibria involving carboxylate groups.

Amide proton titration shifts in H2O solution of bull seminal inhibitor IIA were measured over the pH range from 3 to 6 using two-dimensional correlated spectroscopy. These data enabled characterization of the pKa values for the majority of the carboxylate groups in the protein. Two glutamate side-chains were found to form hydrogen bonds with their own backbone amide proton. Different temperature variations of the populations of these local, cyclic structure elements are indicated for the individual sites.

Site-specific reactivities of cysteine residues in horse L-apoferritin.

Recombinant horse L-apoferritin and its mutants were used to compare the reactivities of two different cysteinyl residues with 7-fluoro-4-sulfamoyl-2,1,3-benzoxadiazole (ABD-F), p-chloromercuribenzoic acid (PCMB), N-(9-acridinyl)maleimide (NAM), and 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (NEM-TEMPO). ABD-F selectively reacted with cysteine 52 (C52), which is located on the inner surface of the peptide shell of apoferritin. In contrast, PCMB reacted only with cysteine 130 (C130), which is located at the 3-fold channels of the shell. NAM and NEM-TEMPO reacted with both C52 and C130.

A protein kinase C with divalent cations contributes to thromboxane A2-induced contraction in rabbit vascular smooth muscle.

We investigated the mechanism of contraction induced by a stable thromboxane A2 receptor agonist, STA2, in rabbit aortic smooth muscles. STA2 induced a long-lasting contraction which persisted for over 5 hours. This contraction was found to be potently inhibited by EDTA. In the presence of EGTA, STA2 was able to slowly contract muscle to a near maximum level, suggestive of an extracellular Ca(2+)-independent component in STA2 action. Inhibition of the STA2-induced contraction by EDTA was partially overcome by the addition of Mg2+. Ca2+ and Mn2+ were also effective in attenuating the inhibition. A phorbol ester, PDBu, an activator of PKC (protein kinase C), induced a long lasting contraction in a manner similar to that of STA2. PKC inhibitors, staurosporine and H-7, inhibited the lasting contractions induced by STA2 and PDBu. PKC inhibitors abolished STA(2)-induced contraction in the absence of extracellular Ca2+, suggesting that Ca(2+)-influx from the extracellular space as well as PKC activation are involved in STA(2)-induced contraction. ML-7, a myosin light chain kinase inhibitor, also inhibited the STA(2)-induced contraction, but it did not abolish the contraction in the absence of extracellular Ca2+. Furthermore, STA2 elicited phosphatidylcholine hydrolysis in cultured aortic smooth muscle cells. From the results obtained, we arrived at the hypothesis that PKC contributes to this lasting contraction in the presence of divalent cations, such as Mg2+, Ca2+ or Mn2+. Of these, Mg2+ is the most capable of maintaining this contraction. The Ca-dependent process alone could not account for the long lasting contraction induced by STA2 in vascular smooth muscles.

Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.

From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.

Natural and artificial carbohydrate-glued protein aggregates.

Carbohydrate gluing (which may have a carbohydrate-lectin binding mechanism) was first recognized as a major contributor in the supramolecular assembly of annelid giant Hb from the marine-worm P. aibuhitensis. Although this assembly obviously also relies on protein-protein interactions, the authors tested the application of carbohydrate gluing in the assembly of a protein aggregate using a lectin and a carbohydrate-containing protein. The resultant aggregate was a mixture of the protein aggregate and the ingredient proteins. The significance of the method is that the assembly of the aggregate can be controlled by using a hapten sugar. This controllability, in conjunction with newly developing glyco-technology, has great potential for the construction of arbitrary protein molecules into a regular protein aggregate, thereby providing sophisticated functions.

Active site structural change of alpha-chymotrypsin due to 2-halogeno-ethanols; comparison with ethanol, I-propanol, and urea.

2-Halogeno-ethanols change the active site structure of alpha-chymotrypsin more rapidly and effectively than ethanol, 1-propanol and urea, probably before producing an extensive conformation change.

2-Halogeno-ethanols as an uncoupler of phosphorylation in rat liver mitochondria.

2-Chloroethanol, 2-bromoethanol and 2,2,2-trifluoroethanol at a concentration of 0.79 vol.% stimulated state 4 respiration and released oligomycin inhibition of state 3 respiration. 2-Fluoroethanol and 1-propanol at the same concentration did not affect the respiration.

Inhibition of alpha-chymotrypsin by 2-halogeno-ethanols; comparisons with 2-methyl-ethanols and urea.

2-Halogeno- and 2-methyl-ethanols inhibit alpha-chymotrypsin in the order of their substituted groups: [1] tri- greater than di- greater than mono-, [2] Br- greater than Cl- greater than CH3- greater than F-. The inhibition by the halogeno-ethanols is mediated differently from that by the methyl-ethanols, ethanol, and urea.

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis.

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.

Thromboxane A2 receptor-mediated signal transduction in rabbit aortic smooth muscle cells.

1. 9,11-Epithio-11,12-methenothromboxane A2 (STA2), a stable analogue of thromboxane A2 (TXA2), contracted rabbit aortic smooth muscles (RASM) and accumulated [3H]inositol phosphates in cultured RASM cells. The contraction and phosphoinositide hydrolysis were competitively inhibited by TXA2 receptor antagonists, including ONO NT-126, S-145, SQ29548, KW3635, GR32191B and ONO3708. 2. STA2 inhibited [3H]ONO NT-126 binding in a concentration-dependent manner in membranes derived from cultured aortic smooth muscle cells, but GTP gamma S, a stable GTP analogue, did not affect STA2-induced inhibition of [3H]ONO NT-126 binding. 3. The time course analysis revealed that STA2 rapidly decreased inositol phosphate level and therefter increased. Pertussis toxin did not attenuate but rather increased STA2-induced phosphoinositide hydrolysis. 4. TXA2 receptor stimulation results in at least two signaling pathways in RASM cells: stimulation and inhibition of phosphoinositide hydrolysis.

EDTA and EGTA can discriminate tonic contractions induced by thromboxane A2 and phorbol ester in rabbit aortic smooth muscles.

The effects of chelating agents of divalent cations on the contraction induced by STA2, a stable thromboxane A2 analog, were examined in rabbit aortic smooth muscles, comparing with a phorbol ester-induced contraction. Pretreatment of muscles with EDTA (4 mM) resulted in potent inhibition of STA2 (1 nM)-induced contraction. However, STA2 could contract muscles slowly in the presence of EGTA (4 mM). The muscles contracted with STA2 relaxed rapidly after addition of EDTA and/or EGTA. However, STA2 recovered contraction in the presence of EGTA, but not of EDTA. PDBu, phorbol 12,13-dibutyrate, also contracted the muscles slowly and potently. Pretreatment with EDTA, but not EGTA, attenuated PDBu-induced contractions. The muscles contracted with PDBu relaxed after the addition of EDTA, but not of EGTA. The present results imply that the vascular smooth muscle contractions are composed of two distinct components: a short-lived contraction that is related to Ca2+ and a lasting contraction that is related to Mg2+, and EDTA and EGTA are good tools for discriminating the tonic phase of STA2- and/or PDBu-induced contraction.

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets.

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.

Kinetic study of CO and O2 binding to horse heart myoglobin reconstituted with synthetic hemes lacking methyl and vinyl side chains.

Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this view.

Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.

Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."