CM-Sephadex Column Research Articles

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications.

We characterized trypsin- and chymotrypsin-like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio-formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76- and 14.05-fold for trypsin and chymotrypsin, respectively. Michaelis-Menten constants (Km ) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ∼21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ∼29 and 34 kDa in SDS-PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio-formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.

Effects of Phospholipase A, Cardiotoxin and Neurotoxin Obtained from Formosan Cobra Venom on the Contractile Responses of Isolated Guinea Pig Ileum

The Formosan cobra venom (Naji naji atra venom, NNAV) was separated into twelve fractions on a column of CMSephadex (C-50) at 4°C with increasing gradients of ammonium acetate buffer (0.005 to 0.9 M) and of pH 5.0-7.0. Fraction 12, cardiotoxin, was further purified via the second CM-Sephadex (C-50) column to get homogeneous peptide. Phospholipase A was found in Fraction 5 to 7 from CM-Sephadex (C-50) column separation. These fractions were then pooled together and further purified by using the method of HClO4 precipitation to produce homogeneous peptide. Neurotoxin was found in Fraction 8. Among the three main components in cobra venom, neurotoxin, cardiotoxin and phospholipase A, only cardiotoxin and phospholipase A caused the stimulatory effect on guinea pig ileum muscle, which showed tachyphylaxis by itself, but not by cross action. Phospholipase A caused significant contraction on guinea pig ileum muscle at 10-8 to 10-7 g/ml; whereas cardiotoxin caused the same extent of contraction at 10-6 to 10-5 g/ml. Cardiotoxin caused ileum muscle contraction at lower concentrations and turned out paralyzing at higher concentrations; however, phospholipase A caused contraction only even at higher concentrations. Therefore, it was phospholipase A from crude Formosan cobra venom that caused ileum muscle contraction at lower concentrations, and cardiotoxins causing paralysis at higher concentrations. The mechanism of phospholipase A causing guinea pig ileum muscle contraction was not due to the enzyme reaction products such as lysolecithin or free fatty acid, but probably due to the membrane-damaging activity. The induction of guinea pig ileum muscle contraction by phospholipase A could be partially blocked by atropine and morphine, and could be potentiated by mipafox, an irreversible acetylcholinesterase inhibitor. The result revealed the involvement of parasympathetic nervous system. In addition, the contraction could almost be blocked by morphine plus dibenzyline. The result revealed the involvement of serotonin release from enterochromaffin cells. The release of histamine from gut mast cells was less important, because histamine blocker, pyribenzamine, had little effect on its response. In summary, phospholipase A-induced guinea pig ileum muscle contraction was due to the release of serotonin. On the other hand, the release of acetylcholine played a very minor role.

CHROMATOGRAPHIC SEPARATION OF THYROID STIMULATING HORMONE FROM LUTEINIZING HORMONE

A new preparative scale procedure is described for the efficient purification of Thyroid stimulating hormone (TSH) from buffalo pituitary glands. The methodology involves the use of hydrophobic interaction chromatography for the separation of TSH from LH which is more abundant than TSH in the pituitary gland. The two hormones were isolated from the pituitary extract by ammonium sulphate precipitation and were then separated on a Phenyl-Sepharose column. The fractions obtained from Phenyl-Sepharose chromatography were assessed using a Uno-Q column on FPLC where enrichment of the TSH in the PS-2 fraction was evident. The final removal of the trace amounts of the LH was done by ion exchange chromatography in tandem on CM-Sephadex and DEAE-Sephacel columns. All the chromatography fractions were assessed in a heterologous direct binding ELISA using bTSH and buLH as the reference hormones and anti ovine TSH beta and anti bovine LH beta as the antisera. The purified fraction (DEAE-0.5 M peak) when checked exhibited no LH immuno reactivity in a western blot against anti bovine LH beta antiserum. Forty two milligrams of a 200 fold purified buTSH per kg wet glands was obtained.

Cathepsin L Plays an Important Role in the Lysosomal Degradation ofL-Lactate Dehydrogenase

A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313.

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48 degrees C and 4.9, respectively. The enzyme was stable up to 60 degrees C and its pH stability was in the range of 5.0-8.0. The Km and Vmax of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, Ea, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a Ki of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a Ki of 9.5 mg/ml. The enzyme was inhibited by Hg2+, Mn2+, Fe3+ and Cu2+ and enhanced by Co2+ and Mg2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate.

Bowman–Birk inhibitors in soybean seed coats

Soybean Bowman–Birk inhibitor (BBI), possessing a molecular weight (MW) of 8 kD, is a known cancer chemopreventative and anticarcinogenic agent Aqueous extracts of soybean seed coats, rigorously screened to remove cotyledons, contained peroxidase along with three unknown proteins each of which possessed an estimated MW of <10 kD when compared with known standards subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because these extracts tested positively for trypsin inhibitor (TI) activity, our objective was to isolate and characterize the low MW inhibitor(s). To eliminate the solubilization of peroxidase from soy seed coats, we applied the classic methodology for BBI isolation which utilizes 60% ethanol. Soybean seed coats, which represent 8% of the whole seed, were defatted, then twice extracted with 60% aqueous ethanol to yield 6.96% sugary solids with 1.56% Kjeldahl nitrogen and a TI activity of 44 mg TI/g solids. Water extracts of the alcohol extracted residues provided an additional 4.55% solids with 2.71% nitrogen and 27 mg TI/g solids. The protein components in the 60% ethanol solubles were further concentrated by acetone precipitation. The acetone precipitates and water extractables were each fractionated on a CM Sephadex C-25 column eluted with a salt gradient. The consequent column fractions from the acetone precipitates when subjected to SDS-PAGE yielded three proteins, one of which possessed the mobility similar to a commercially available soybean BBI. Because BBI can simultaneously inhibit both trypsin and chymotrypsin, we verified the presence of BBI in those column fractions that eluted after 0.21M NaCl concentration based on colorimetric assays for TI and chymotrypsin inhibitor (CTI) activities. Chymotrypsin negative stain technique applied to native PAGE of the TI and CTI-active fraction from acetone precipitates showed evidence for seven isoforms of BBI. Two CM Sephadex column fractions eluting prior to 0.21 M salt concentration showed two low MW proteins (<7 kD) when stained for protein and compared with MW standards subjected to SDS-PAGE. Based on the summation of CTI active components in the CM Sephadex C-25 column fractions from 60% ethanol extract, their CTI activity relative to CTI in a commercial BBI standard was 61.9%, whereas, the CTI activity in fractions of column chromatographed water extracts that eluted after 0.21M salt concentration was 86.1%. Soybean seed coats may provide a new, inexpensive source of BBI concentrate for use as a potential anticancer agent.

Microheterogeneity in Buffalo Thyroid Stimulating Hormone

Microheterogeneity has been observed in the thyrotropin isolated from buffalo pituitary glands using the standard isolation and fractionation procedures. Crude thyrotropin was passed through CM-Sephadex column for enrichment, followed by sizing through Sephadex G-200 and ultimately DEAE-Sephacel was used for the segregation of TSH from LH. The buTSH obtained had 72% TSH immunoreactivity and a specific bioactivity of 71U/mg. It had only about 10% LH content in it. N-terminal analysis of buTSH revealed heterogeneity where the two N-termini were determined as Phe and Gly. The carbohydrate analysis of the buTSH fraction revealed the presence of three microheterogenous forms, out of which the form containing the biantennary carbohydrate structure had the highest immunoreactivity. However, buffalo TSH was found to be different from sheep TSH. The latter appears to have a high mannose/hybrid carbohydrate structure in the most immunoreactive fraction.

Qualitative change in mice liver HMG proteins after low dose chronic administration of aqueous extract of betel nut and diethylnitrosamine

High mobility group (HMG) proteins have roles in the organization and function of chromatin. Their involvement in carcinogenesis is, however, not clear. Swiss albino mice were exposed to aqueous extract of betel nut (AEBN) and a hepatocarcinogen, diethylnitrosamine (DEN), in a chronic low dose protocol for up to 4 weeks. Studies of their effects on the liver HMG proteins during the process showed that major HMG proteins 1, 2 14 and 17 were qualitatively affected. HMG proteins of the treated animals were found to elute earlier from CM-sephadex column than that of the control. Conformational changes in the HMG proteins under the influence of these carcinogens were observed. The results obtained in this investigation indicate that both carcinogens induced similar changes in HMG proteins of liver of mice.

In Vitro Activity of Sorgum Seed Antifungal Proteins against Grain Mold Pathogens

Several sorghum seed antifungal proteins (AFP), including sormatin, chitinase, glucanase, and ribosome-inhibiting protein (RIP), were extracted, partially purified using sequential ammonium sulfate precipitation, and eluted from a CM-Sephadex column using a linear salt gradient. Eluted fractions were tested for bioactivity against Fusarium moniliforme, Curvularia lunata, and Aspergillus flavus using hyphal rupture, hyphal extension, and spore germination methods. A fraction containing several AFPs was most inhibitory against F. moniliforme, C. lunata, and A. flavus. Spore germination of all three species was markedly inhibited by 360 ppm of AFPs. The inhibitory effects were not observed when the protein fraction was boiled, suggesting the involvement of undenatured proteins. Hyphal elongation inhibition was the least sensitive assay to detect the inhibitory effects of AFPs on these fungi. F. moniliforme exhibited hyphal rupture at the growing tip at protein levels as low as 70 ppm. C. lunata required higher protein levels (70−360 ppm) and ruptured only at hyphal tips. A. flavus did not exhibit hyphal disruption when treated with AFPs. Keywords: Antifungal proteins; sormatin; chitinase; glucanase; sorghum; Fusarium moniliforme; Curvularia lunata; bioactivity

Polyamine oxidase from leaves of Lilium longiflorum: Purification and properties

Summary Polyamine oxidase was purified to homogeneity from leaves of Lilium longiflorum using acetone powder, CM-Sephadex and hexamethylenediamine-Sepharose 4B column chromatography. The enzyme showed optimal pH of 5.5 and Km values of 16 and 6μmol · L−1 with spermidine and spermine, respectively. The enzyme was a monomer with molecular mass of 63,000 u (Da) as judged by sodium dodecyl sulfate Polyacrylamide gel electrophoresis. The enzyme appeared to contain 1 mol of flavin adenine dinucleotide per 63,000 g of the enzyme. Hexamethylenediamine was a potent competitive inhibitor with ki of 7 μmol · L−1. One mol per liter NaCl increased Vmax of the purified enzyme, similar to polyamine oxidase from oats, and also caused an increase of Km. Antibodies raised against the purified enzyme from Lilium longiflorum did not cross-react with polyamine oxidase from Avena sativa, Eichhonia crassipes, Gibasis pellucida, or Zea mays.

Application of thermostable xylanases from Humicola sp. for pulp improvement

Thermophilic Humicola sp. secreted thermostable xylanases when grown on wheat bran medium at 50°C. DEAE-Sephadex A-50 column chromatography of the crude xylanase separated three fractions of xylanase (I, II and III), xylanase I being homogeneous in polyacrylamide gel electrophoresis after CM-Sephadex column chromatography. The respective xylanases, including the crude xylanase, increased pulp brightness but xylanases II and III decreased the viscosity of the pulp due to CMCase activity. The crude xylanase contained lower CMCase activity than xylanases II and III.

Purification and characterization of alkaline serine protease from an alkalophilic Streptomyces sp.

SAP, an extracellular alkaline serine protease produced by Streptomyces sp. YSA-130, was purified to homogeneity by CM-Sephadex column chromatography and crystallization. The enzyme was a monomeric protein with a molecular weight of 19,000 as estimated by SDS-PAGE and gel filtration. The amino acid composition and amino-terminal sequence of SAP were similar to those of other bacterial serine proteases, i.e., Streptomyces griseus proteases A and B, Lysobacter enzymogenes alpha-lytic protease and Nocardiopsis dassonvillei subsp. prasina OPC-210 alkaline serine protease NDP-I. The optimum temperature and pH for the enzyme activity were 60 degrees C and 11.5. The enzyme was stable up 50 degrees C, and between pHs 4 and 12. The activity was inhibited by Ag+, Hg2+, Co2+, sodium dodecyl sulfate. N-bromosuccinimide, diisopropyl phosphorofluoridate (DFP), 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), iodoacetate, N-ethylmaleimide (NEM), phenylmethanesulfonyl fluoride (PMSF), and phenylglyoxal.

Characterization of a Zn(2+)-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity.

p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.

One-step preparation of Hageman factor fragment concentrate

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM-Sephadex column. The preparation was free of the main kallikrein inhibitors--Cl-inactivator, alpha 2-macroglobulin, antithrombin III and alpha 1-antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.

Regulation of rat hepatic peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme by thyroid hormone

Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.

Purification and properties of ascorbate oxidase from cucumber fruit

Abstract Ascorbate oxidase [AOD, EC 1.10.3.3], a copper-containing enzyme, was purified to homogeneity from cucumber fruit (Cucumis sativus) by CM-Sephadex and hydroxyapatite column chromatography and chromatofocusing. Isoelectric point (pI) of the purified enzyme was 8.3. Two other isoforms of AOD with alkaline pI values were also found. Purified AOD (MW 150,000) was a glycoprotein composed of two identical subunits (MW 70,000). N-terminal amino acid sequence was determined as Gly-Phe-Pro-Lys-Ile-Lys-His-Tyr-Lys-Trp-Asp-Val-Glu-Tyr-Met-Phe.

Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase.

The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory gamma subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine gamma subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of lambda phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the lambda cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue gamma subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of gamma in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic gamma with the same amino acid sequence as that of native gamma. Both fusion protein and synthetic gamma inhibited trypsin-activated phosphodiesterase with high affinity (Kd less than 100 pM). Likewise, both were as effective as native gamma in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of gamma is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of gamma stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of gamma is critical for inhibition of the phosphodiesterase.

Two Apparent Molecular Forms of Bovine Lactoferrin

Bovine lactoferrin was prepared by CM-Sephadex column chromatography from defatted colostrum. When partially purified lactoferrin was analyzed by SDS-PAGE apparently two polypeptides of different size appeared. The polypeptides were transferred to a nitrocellulose sheet and visualized using antirabbit serum raised against the small polypeptide. Two polypeptides appeared clearly when stained by an immunological method. The color intensity of the two polypeptides was similar when the polypeptides were stained with Coomassie Brilliant Blue R-250. A high similarity of cyanogen bromide cleavage patterns between the two polypeptides was also observed in those stained with dye and immunologically visualized. Therefore, it is suggested that these polypeptides are lactoferrins. Comparison of the peptide patterns of the two lactoferrin molecules being deglycosylated suggested that sugar moieties may be one, but not all, of the causes of the size heterogeneity in lactoferrin. These results suggest that bovine colostrum contains two lactoferrin molecules of different size, although the physiological significance of the heterogeneity is not yet known.

Heterogeneity of the bovine adrenal steroid 21-hydroxylase.

The results presented indicate that purified cytochrome P-45021 which migrated upon SDS gel electrophoresis essentially as a single band, is further separable into different species by ion-exchange chromatography. The P-450 eluted from the CM-Sephadex column at different points along the buffer concentration gradient, exhibited significant differences in (1) the 21-hydroxylation of 17 alpha-OH-progesterone compared to progesterone and (2) the Type I spectral change produced by 17 alpha-OH-progesterone compared to that due to delta 4-androstenedione. These results indicate that the purified P-450 which appeared homogeneous contains different species differing in net charge and steroid preferences for 21-hydroxylation and binding. The ratio, 21-hydroxylation of 17 alpha-OH-progesterone/progesterone ranged between 2.6 and 0.86 suggesting that the purified preparation is a mixture of 17 alpha-OH-progesterone preferring and progesterone preferring species. Possible molecular bases for the heterogeneity of the 21-hydroxylase are discussed.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.