Purification of a 41 kDa cod-allergenic protein

Cod fish is one of the foods most frequently involved in allergy. Only the cod allergen Gad c I, a 12.3 kDa parvalbumin, has been purified and characterized. Recently, we have detected allergen bands which have not previously been described, in particular a 41 kDa protein, by Western-blot. In the present work, this protein has been purified from a crude cod extract by ammonium sulfate fractionation, hydroxyapatite chromatography and preparative electrophoresis; a single band with an M_r of 41×⁻³ was found in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition and the isoelectric point of the protein were determined. The purified protein (p41) was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals and to a monoclonal anti-parvalbumin which recognizes specifically the first calcium binding site of parvalbumins. p41 may therefore contain a calcium binding site corresponding to an IgE-epitope similar to that of Gad c I.     

Purification and partial amino acid sequence of a 28 kDa cyclophilin-like component of the rat liver sigma receptor

The sigma (σ) receptor, a putative non-opioid receptor site which has been suggested to function as a neuromodulator of dopaminergic and NMDA systems, and is found in brain, liver and many other tissues, has been purified > 560-fold from a detergent-solubilized rat liver membrane preparation by affinity chromatography, using an affinity matrix prepared from an oximino derivative of haloperidol. The affinity column selectively retained principal components of M_r 28 kDa, 40 kDa and 65 kDa that could be eluted from the column with σ-selective ligands, specifically dextrallorphan and haloperidol. After dialysis and concentration by ultrafiltration, a loss in density of the 65 kDa component and an increase in the 28 kDa and 40 kDa components was observed. A 15 amino acid N-terminal sequence was obtained for the 28 kDa protein which is identical to the N-terminal sequence of the 17 kDa rat cyclophilin A, a cytosolic protein, suggesting that a critical component of the rat liver σ receptor may be a cyclophilin. These results support the suggestion that σ receptors are a key link between the central nervous system and the immune system.     

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.     

Purification and partial peptide sequence analysis of the boar 32 kDa sperminogen

Boar 32 kDa sperminogen was purified from acid extracts of washed epididymal spermatozoa, and partial peptide sequence was determined. Boar sperminogen was purified from the acid extracts of boar spermatozoa by gel filtration through Sephadex G-75 column, followed by preparative SDS-PAGE. Gelatin zymographic analysis of the gel-filtered fractions showed that sperminogen was composed of three separate proteolytic bands. Among the three proteolytic bands, the 32 kDa sperminogen band which showed the strongest proteolytic activities upon activation was sliced out and eluted from the gel fragments. The eluted 32 kDa sperminogen was then subjected to peptide sequencing. Since the N-terminus of the 32 kDa sperminogen was blocked for peptide sequencing by Edman degradation method, the internal amino acid sequence of the sperminogen was obtained from the CNBr-digested peptides of sperminogen. The amino acid sequence of the analyzed peptide of the 32 kDa sperminogen showed 100% identity with that of proacrosin.     

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47-9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.     

Purification of 52 kDa protein: a putative component of the import machinery for the mitochondrial protein-precursor in rat liver

A protein having a molecular mass of 52 kDa was purified to homogeneity from solubilized mitochondrial membrane proteins by affinity column chromatography using the synthetic presequence of ornithine aminotransferase (OAT) as the ligand. This 52 kDa protein was specifically bound to the affinity column and eluted with 1 mM OAT-presequence, indicating that it recognized the presequence and bound to it specifically. Anti-52 kDa protein Fab fragments specifically inhibited the import of OAT-precursor into mitochondria, showing that the 52 kDa protein plays an essential role in this process. These results suggest that 52 kDa protein is a component of the import machinery of the mitochondrial protein-precursor in the mitochondrial membrane.     

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757–767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A–500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS–PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc₅), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc_n, n=2–6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the β anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with K_i values depending on the pH of the reaction mixture (K_i=0.013−0.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin (k_cat/K_m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/K_m) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as K_m, k_cat and k_cat/K_m successively.     

Chicken gizzard 5'-nucleotidase is a receptor for the extracellular matrix component fibronectin

The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified enzyme was reconstituted into 3H-labeled proteoliposomes which were used as a model to study the association of a membrane protein with fibronectin. We demonstrated that the binding process between proteoliposomes and fibronectin has the qualities of a receptor-ligand interaction, i.e., is saturable and specific. In contrast to the association of fibronectin with integrins, the interaction with 5'-nucleotidase does not require divalent metal ions. Synthetic peptides containing the RGD-sequence or a monoclonal antibody interfering with binding of other receptors to the cell-binding domain of fibronectin did not abolish the interaction with 5'-nucleotidase. This indicates that the RGDS-sequence does not represent the major contact site for the AMPase and that the 5'-nucleotidase belongs to a separate class of fibronectin receptors with distinct properties as compared to the integrins.     

细脚拟青霉菌丝体多糖纯化及组成分析

通过DE-52纤维素柱和Sephedex G-100葡聚糖凝胶柱,对细脚拟青霉Paecilomyces tenuipes菌丝体粗多糖进行纯化,得到PtPs1和PtPs2,应用离子色谱HPAEC-PAD对纯多糖PtPs1和PtPs2的单糖组成进行分析,证实均由葡萄糖、半乳糖和甘露糖三种单糖组成,经红外扫描确定PtPs1和PtPs2均为α型吡喃糖。     

Purification and characterization of a 24 kDa protein from tartary buckwheat seeds

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.     

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.     

Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii

An exocellular proteinase synthesized by the geophilic dermatophyte Trichophyton vanbreuseghemii has been purified and characterized. The fungus obtained from soil in Iran was cultivated in modified Czapek–Dox liquid medium containing 0.1% bacteriological peptone and 1% glucose as the nitrogen and carbon sources. Partial purification of the proteinase was accomplished by (NH₄)₂SO₄ precipitation, followed by ion exchange chromatography. Analysis of the enzyme by SDS-PAGE revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Proteinase activity was optimum at pH 8, but remained high in the range of pH 7–11. Moreover, the partially purified enzyme presented a keratinolytic activity as evidenced by the keratin azure test. The inhibition profile and the good activity of the enzyme towards the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide suggested that it belonged to the chymotrypsin/subtilisin group of serine proteinases. The keratinolytic properties of T. vanbreuseghemii suggest that this fungus may be an alternative for the recycling of industrial keratinic wastes.     

Purification and characterization of a lipocortin-like 33 kDa protein from guinea pig neutrophils

A lipocortin-like, phospholipase A2 inhibitory 33 kDa protein was purified from guinea pig neutrophils. From amino acid composition and sequence data, this protein was found to have a high degree of homology to human lipocortin I. This protein inhibited porcine pancreatic phospholipase A2 activity in the presence of [3H]oleic acid-labeled Escherichia coli membranes as substrate. Maximal inhibition amounted to 65% whereas 50% inhibition occurred at 83.5 nM. This protein showed F-actin-binding ability in a Ca2+-dependent manner.     

Purification and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus

The molecular mass of esterases usually falls in the range of 20–160 kDa, although an esterase of 5.7 kDa from Candida lipolytica has been described. Three other enzymes smaller than 10 kDa have been reported, all of which were more thermostable than their higher molecular mass counterparts. This paper describes the purification of an extracellular esterase hydrolysing fluorescein dibutyrate from Bacillus stearothermophilus NCIMB 13335. The esterase had a molecular mass of 1.57 kDa when analysed by SDS-PAGE, gel filtration and MALDI-TOF spectrometry. This enzyme retained more than 90% of its activity after incubation at 90°C for 2 h.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Examination of a nerve injury-induced, 37 kDa protein: Purification and characterization

Following traumatic injury to the adult rat sciatic nerve the synthesis and accumulation of soluble, extra-cellular, 37 kDa protein is increased. This protein, which accumulates in the extracellular space of the injured nerve, accounts for nearly 5% of the total soluble pool of protein in an injured nerve 3 weeks after injury. 8 weeks after injury, when regeneration is nearly complete, this accumulated pool returns to control levels, yet if regeneration is blocked synthesis of the 37 kDa protein remains high. Recently this 37 kDa protein has been shown to be nearly identical to apolipoprotein E, the protein component of various lipoprotein particles. This finding suggests a role for the 37 kDa protein in cholesterol and lipid transport and metabolism during nerve repair within the nervous system, functions that have been ascribed to apo E in serum. Results are presented here describing the purification of the nerve injury induced 37 kDa protein and the subsequent production of specific rabbit antisera directed against it. By centrifugation analysis in a sucrose gradient, a native mass of 37 kDa was determined, revealing the 37 kDa protein's monomeric, native structure. Additionally injections of [³⁵S]methionine directly into the injured nerve allowed 1) a comparison of 37 kDa synthesis in vivo versus in vitro and 2) an examination of the presence or absence of retrogradely transported 37 kDa protein. The in vitro and in vivo collected material were found to share identical 2-dimensional electrophoretic mobilities, and no appreciable amount of transported 37 kDa protein was found in proximal regions of the injured nerve.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

A 25 kDa alpha 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase.

Besides the monomeric mammalian 95 kDa progelatinase, two additional forms, a disulfide-bridged 220 kDa dimer and a 125 kDa form were isolated from human PMN leukocytes. The 125 kDa progelatinase was identified as a covalently linked, disulfide-bridged heterodimer formed of the monomer with a 25 kDa protein. This 25 kDa protein was isolated from gelatinase bound to the affinity support of gelatin-Sepharose and eluted by DTE-containing buffer. The amino acid sequence of tryptic peptides of this protein revealed homology with an alpha 2-microglobulin-related protein from rats, a protein so far unknown in humans.