Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim

A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS–PAGE, with an apparent molecular mass of 13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC₅₀ of 1.6 ng/ml. Liquid chromatography–tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.     

Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luffa acutangula

A glycoprotein with a molecular weight of 28,000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.     

Improved isolation and further characterization of beta-trichosanthin,a ribosome-inactivating and abortifacient protein from tubers of trichosanthes cucumeroides (cucurbitaceae)

• 1.1. Beta-trichosanthin was isolated from root tubers of Trichosanthes cucumeroides with a procedure involving acetone fractionation, ion exchange chromatography on CM-Sepharose and DEAE-Sepharose and gel filtration on Sephadex G-50.
• 2.2. The protein was homogeneous by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. It possessed a molecular weight of 28,000 and was a strongly basic glycoprotein.
• 3.3. It was immunochemically identical to trichosanthin but different from alpha- and beta-momorcharins.
• 4.4. It possessed potent abortifacient and ribosome-inactivating activities. In the latter type of activity it was more potent than trichosanthin.

     

Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity

Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912).     

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.     

Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Purification and characterization of novel calmodulin-binding protein from cardiac muscle

A novel protein which represents the most abundant calmodulin-binding protein in bovine heart cytosolic fraction was purified to apparent homogeneity. The purification procedure involved DEAE-Sepharose CL-6B (to remove calmodulin), calmodulin-Sepharose 4B affinity, and Sepharose 6B column chromatographies. This purified calmodulin-binding protein is a highly asymmetric protein with a sedimentation coefficient of approximately 5.0 S and a Stokes radius of about 83.0 A. The molecular weight of the calmodulin-binding protein was determined to be 175,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein showed a single protein band with an apparent molecular weight of 140,000. The result suggests that the protein is monomeric. Although this molecular weight is similar to that of caldesmon, a known ubiquitous calmodulin-binding protein, the protein did not react with caldesmon-specific antibodies, nor did it display a proteolytic fragmentation pattern similar to that of the former. In addition, caldesmon was found almost exclusively in the particulate fraction in low ionic strength cardiac muscle extract, whereas this protein is purified the soluble fraction.     

Purification and characterization of four isoforms of Himalayan mistletoe ribosome-inactivating protein from Viscum album having unique sugar affinity

Ribosome-inactivating proteins having antitumor and immunomodulatory properties constitute the active principle of widely used mistletoe therapy in Europe. This is the first report of the four isoforms of Himalayan mistletoe ribosome-inactivating proteins (HmRips) from Viscum album parasitized on wild apple inhabiting NW Himalayas. HmRips were purified by affinity chromatography and four isoforms were separated by ion-exchange chromatography. HmRip 1, 2, 3, and 4 have isoelectric points of 6.6, 6.1, 5.2, and 4.7, respectively. Disulfide linked toxin and lectin subunits of HmRip 1 and 2 isoforms have molecular weights of 28 and 34kDa while those of HmRip 3 and 4 have 28 and 32kDa. The isoforms lacked blood group specificity and showed positive activity with seven mammalian erythrocyte types but did not show any activity with avian erythrocyte type. Lectin activity of HmRips remained unchanged for a wide range of temperatures (0-65 degrees C) and pH (3-9). Unlike other type II Rips, the HmRip 1, 2, and 4 showed unique affinity towards l-rhamnose, meso-inositol, and l-arabinose while HmRip 3 has specificity to gal/galNAc. Sugar binding studies with 22 sugars also suggested that the C-4 hydroxyl of galactose might be the critical site involved in sugar binding of HmRips. Type II Rips are known to be galactoside specific and do not have affinity for l-rhamnose and meso-inositol. However, HmRip 1, 2, and 4 having equal affinity for galactose and l-rhamnose do not strictly fit into any of the four structural classes of the lectins and represent a new class of type II Rips and plant lectins.     

Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.     

Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds

The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.     

Purification and characterization of a silica-induced bronchoalveolar lavage protein with fibroblast growth-promoting activity

Experimentally induced silicosis provides a good model for chronic interstitial pulmonary inflammation and fibrosis. In the present study, a specific single polypeptide with an apparent molecular mass of 58,000 and a pI of 4.5 was purified and characterized from the bronchoalveolar lavage fluid of silicotic rats. The same protein was also isolated from both the extract and conditioned medium of alveolar macrophages of silicotic rats. Therefore, this protein was termed an inducible silicotic (rat) bronchoalveolar lavage protein-p⁵⁸ (iSBLP⁵⁸) or an inducible silicotic (rat) pulmonary macrophage factor (iSPMF-p⁵⁸). iSBLP⁵⁸ has been purified to homogeneity by a combination of gel permeation, Mono Q ion exchange, and reverse-phase high performance liquid chromatography. This polypeptide displayed a potent fibroblast growth-promoting activity in vitro. The sequence of the first 15 NH₂-terminal amino acids was determined and was found to have high sequence homology with members of the mammalian chitinase-like protein family, which includes human cartilage gp39, mammalian oviduct-specific glycoprotein, and a secretory protein from activated mouse macrophages. J. Cell. Biochem. 67:257–264, 1997. © 1997 Wiley-Liss, Inc.     

Purification and characterization of a novel calcium-dependent protein kinase from soybean

A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A.C., Putnam-Evans, C.L., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and Blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 microM free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2(+)-ATP of 8 microM and a Vmax of 1.7 mumol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2(+)-GTP, in addition to Mg2(+)-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.     

Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane

Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.     

Purification and characterization of a novel human red cell enzyme with diphenol oxidase activity

A new enzyme protein, diphenol oxidase, has been isolated and purified from human red cells and catalyzes the in vitro formation of adrenochrome from epinephrine and of melanin from dihydroxyphenylalanine. Some immunological and physicochemical properties of this protein have been studied.     

Purification and characterization of a novel 70-kDa brain protein associated with seizure activities

Using ion exchange HPLC and ammonium sulfate precipitation, we have purified a 70-kDa protein (P70) specific to the cobalt-induced epileptogenic cortex of rat cerebrum and determined certain of its biochemical properties. P70 has a similar isoelectric point (pI; 4.6–4.8), amino acid composition and N-terminal amino acid sequence to rat serum albumin (RSA). Intracortical application of purified P70 to the motor area of normal rat cerebrum induces both ECoG seizure discharges and behavioral seizures. The data suggest that P70 is a novel albumin-like protein linked to the generation of seizure activities. However, it can be clearly distinguished from RSA, since it is able to produce seizure, is a glycoprotein and can be readily separated from RSA by 2-dimensional electrophoresis.     

A novel retinol-binding protein from rat. Purification and partial characterization

A novel retinol-binding protein, resolved during purification into two essentially identical forms, has been discovered in the rat. It was purified to apparent homogeneity, using whole neonatal rat pups as source. The protein is distinct from other known retinol-binding proteins by behavior during purification, spectra of bound retinol, and immunochemical reactivity. It is a single polypeptide chain with molecular weight of about 16,000. The protein binds all-trans-retinol as an endogenous ligand. Retinol bound to the protein exhibited considerably altered absorbance and fluorescence excitation spectra compared to free retinol in organic solvent. The retinol-binding protein was found by radioimmunoassay in a number of tissues of the neonatal rat. However, liver and intestine had levels 100-fold higher than any other tissues examined. The intestine of the adult rat had levels 500-fold higher than any other tissue examined, with a decreasing gradient from jejenum to colon. The high levels in intestine suggest this protein may have a role in the absorption of retinol.     

Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.     

Purification and characterization of multiplication-stimulating activity (MSA) carrier protein

The rat liver cell line, BRL-3A, is known to produce a family of polypeptides referred to as multiplication-stimulating-activity (MSA). Serum-free conditioned medium from this cell line is a rich source for the purification of these somatomedin-like molecules. Somatomedins in serum, as well as MSA produced by BRL-3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000–70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.     

Purification and characterization of an endotoxin-binding protein with protease inhibitory activity from Limulus amebocytes

Using a lipopolysaccharide affinity column and ion exchange chromatography, a 12-kDa protein has been purified from Limulus amebocytes. In solid phase binding assays, the radiolabeled protein binds specifically to lipopolysaccharide (LPS) with a Kd value on the order of 10(-7) M. A cDNA coding for this protein has been isolated and sequenced. The amino acid sequence deduced from the cDNA indicates that this protein shares no sequence homology with LPS-binding proteins isolated from different species of vertebrates (Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., and Ulevitch, R. J. (1990) Science 249, 1429-1431) and invertebrates (Aketagawa, J., Miyata, T., Ohtsubo, S., Nakamura, T., Morita, T., Hayashida, H., Miyata, T., Iwanaga, S., Takao, T., and Shimonishi, Y. (1986) J. Biol. Chem. 261, 7357-7365). The binding to LPS can be displaced by the unlabeled 12-kDa protein, polymyxin B, lipid A, and to a lesser extent by D-glucosamine. In whole cell binding assays, the 12-kDa protein has also been shown to bind to Escherichia coli. Using both [14C]casein and a synthetic substrate, the protein has been shown to inhibit the proteolytic activity of trypsin, with an IC50 of approximately 10(-7) M. In the presence of LPS, the antitryptic acitivity of the Limulus endotoxin-binding protein-protease inhibitor remains unaffected. The protein is a major component of the cytoplasmic proteins (1%). Immunocytochemical analysis reveals that this protein exists in the secretory granules of the amebocytes where enzymes and substrates for the clotting cascade reside. Based on the unusual dual functional properties, the newly isolated protein was named a "Limulus endotoxin-binding protein-protease inhibitor" (LEBP-PI).