Purification and characterization of two β-glucosidases from a thermo-tolerant yeast Pichia etchellsii

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible β-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0–9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl β-d-glucopyranoside and alkyl β-d-glucosides whereas BGLII was most active on cellobiose. k_cat and K_M values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl-glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C₁₀) using dimethyl sulfoxide stabilized single-phase reaction microenvironment. About 12% molar yield of octyl-glucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned β-glucosidases from this yeast.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae.

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae

We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic characteristics, such as thermo-denaturation kinetics and dependencies on pH and temperature, but quite different substrate specificities: BGLI hydrolyzed cellobiose efficiently, but BGLII did not. This result is consistent with the observation that the S. cerevisiae transformant carrying BGL1 fermented cellobiose to ethanol but the transformant carrying BGL2 did not. Southern blot analysis revealed that the two beta-glucosidase genes were derived from Saccharomycopsis fibuligera and that the nucleotide sequences of the two genes are closely related. The complete nucleotide sequences of the two genes were determined. BGL1 and BGL2 encode 876- and 880-amino-acid proteins which were shown to be highly similar to each other. The putative precursors begin with hydrophobic segments that presumably act as signal sequences for secretion. Amino acid analysis of the purified proteins confirmed that BGL1 and BGL2 encode BGLI and BGLII, respectively.     

Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a thermo-tolerant yeast

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C _m, occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T _m, (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

Characterization of a β-Glucosidase Produced by a High-Specific Growth-Rate Mutant of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis>

The mutant strain PN-120 of Cellulomonas flavigena produces a ss-glucosidase that is 10-fold more active than the corresponding enzyme isolated from the parental strain. These enzymes were partially purified through Q Sepharose and Bio-Gel filtration. A single protein band was detected on polyacrylamide-gel electrophoresis/zymogram using 4-methylumbelliferyl-beta-D-glucoside. On sodium dodecyl sulfate-PAGE, the enzyme displayed three protein bands, suggesting that in C. flavigena the enzyme is oligomeric with a molecular mass of 210 kDa. On purification, the specific activity of ss-glucosidase isolated from PN-120 was increased 16-fold and showed three times more affinity for cellobiose than the enzyme of the parental strain; nevertheless, the optimum pH and temperature were similar for both enzymes. The kinetic parameters suggested that the increase in the activity of the enzyme, from the mutant strain, was caused by a mutation that affects the catalytic site of the enzyme. The partial amino-acid sequence of the isolated enzyme confirmed that it is a beta-glucosidase because of its homology with other beta-glucosidases produced by cellulolytic bacteria and fungi.     

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.     

Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

Cloning and functional expression of thermostable β-glucosidase gene from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis>

A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.     

β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance

From Aspergillus tubingensis CBS 643.92 four distinct beta-glucosidases (I-IV) were purified by a four-step purification procedure. SDS-PAGE revealed molecular masses of 131, 126, 54 and 54 kDa, respectively, and their isoelectric points were determined to be 4.2, 3.9, 3.7 and 3.6, respectively. The beta-glucosidases exhibited high diversity with respect to pH and temperature optima and stability, as well as to substrate specificity and glucose tolerance. The major beta-glucosidase (I) preferentially hydrolysed oligosaccharides. The acid-stable and heat-tolerant beta-glucosidase II hydrolysed aryl and terpenyl beta-D-glucosides as well as 1-O-trans-cinnamoyl beta-D-glucoside. In contrast to beta-glucosidases I and II, the minor beta-glucosidases III and IV were found to be glucose-tolerant; inhibition constants of 470 and 600 mM, respectively, were determined.     

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate.

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.     

alpha-L-Arabinofuranosidase from Streptomyces sp. PC22: purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues

alpha-l-Arabinofuranosidase was purified from culture filtrates of the thermoalkaliphilic Streptomyces sp. PC22 to about 108-fold purity by (NH(4))(2)SO(4) precipitation followed by column chromatography. Its approximate molecular weight was 404kDa, with a subunit mass of approximately 79kDa. The evaluated K(m) and V(max) values with p-nitrophenyl-alpha-l-arabinofuranoside as substrate were 0.23mM and 124 U.mg(-1), respectively. The purified enzyme was optimally active at 65 degrees C and pH 6.0 and showed a mild but significant synergistic effect in combination with other xylanolytic enzymes, including xylanase, beta-xylosidase and acetyl esterase, on the degradation of oat-spelt xylan, corn cob and corn husk substrates with a 1.25, 1.32 and 1.21-fold increase in the amount of reducing sugar released, respectively, compared to the expected (additive) amounts for the individual enzymes acting alone. Sequential reactions using two xylan-backbone degrading enzymes (xylanase/beta-xylosidase) and two debranching enzymes (alpha-l-arabinofuranosidase/acetyl esterase) were also determined. The highest degree of synergy was obtained in sequential reactions with the debranching enzyme digestion preceding the xylan-backbone degrading enzymes.     

Purification and properties of mouse liver coproporphyrinogen oxidase

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.     

Isolation and characterization of a thermostable intracellular enzyme with peroxidase activity from Bacillus sphaericus

During a screening for bacteria producing enzymes with peroxidase activity, a Bacillus sphaericus strain was isolated. This strain was found to contain an intracellular enzyme with peroxidase activity. The native enzyme had a molecular mass of above 300 kDa and precipitated at a salt concentration higher than 0.1 M. Proteolytic digestion with trypsin reduced the molecular mass of the active enzyme to 13 kDa (dimer) or 26 kDa (tetramer) and increased its solubility, allowing purification to homogeneity. Spectroscopic investigations showed the enzyme to be a hemoenzyme containing heme c as the covalently bound prosthetic group. The enzyme was stable up to 90 degrees C and at alkaline conditions up to pH 11, with a pH optimum at pH 8.5. It could be visualized by activity staining after SDS-PAGE and showed activity with a number of typical substrates for peroxidases, e.g., 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt, guaiacol and 2,4-dichlorophenol; however the enzyme had no catalase and cytochrome c peroxidase activity.     

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of beta-glucosidases in a high throughput fashion.     

Microbial beta-glucosidases: cloning,properties, and applications

Beta-glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.     

Purification and characterization of an esterase hydrolyzing monoalkyl phthalates from Micrococcus sp. YGJ1

An esterase that specifically hydrolyzes medium-chain (C(3)-C(5)) monoalkyl phthalates was purified from phthalate-grown Micrococcus sp. YGJ1. The enzyme activity was split into two fractions by hydrophobic chromatography on Phenyl Sepharose, and the enzymes were purified to homogeneity from each fraction. The purified enzymes showed similar properties with respect to molecular mass (60 kDa), subunit molecular mass (27 kDa), N-terminal amino acid sequence, optimal pH (about 7.5), temperature-dependence, substrate specificity, and inhibitor susceptibility. The enzymes showed no activity toward various dialkyl phthalates or aliphatic carboxyl esters. 2-Mercaptoethanol effectively protected the enzymes from spontaneous inactivation. Diethylpyrocarbonate, p-chloromercuribenzoate, Hg(2+), and Cu(2+) strongly inhibited the enzymes, while phenylmethylsulfonyl fluoride produced weak inhibition, and various metal chelating reagents were ineffective. These findings show that the enzymes bear a close resemblance to the putative phthalate ester hydrolase (PehA) of Arthrobacter keyseri 12B.     

Some structural properties of plant serine:glyoxylate aminotransferase?

The structural properties of photorespiratory serine:glyoxylate aminotransferases (SGAT, EC 2.6.1.45) from maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves were examined. By means of molecular sieving on Zorbax SE-250 column and filtration through centrifugal filters it was shown that dimers of wheat enzyme (molecular mass of about 90 kDa) dissociate into component monomers (molecular mass of about 45 kDa) upon decrease in pH value (from 9.1 or 7.0 to 6.5). At pH 9.1 a 50-fold decrease of ionic strength elicited a similar effect. Under the same conditions homodimers of the maize enzyme (molecular mass similar to that of the wheat enzyme) remained stable. Immunoblot analysis with polyclonal antiserum against wheat seedling SGAT on leaf homogenates or highly purified preparations of both enzymes showed that the immunogenic portions of the wheat enzyme are divergent from those of the maize enzyme. The sequence of 136 amino acids of the maize enzyme and 78 amino acids of the wheat enzyme was established by tandem mass spectrometry with time of flight analyzer. The two enzymes likely share similarity in tertiary and quaternary structures as well as high level of hydrophobicity on their molecular surfaces. They likely differ in the mechanism of transport from the site of biosynthesis to peroxisomes as well as in some aspects of secondary structure.     

Phenylalanyl-tRNA synthetases from sheep liver and yeast. Correlation between net charge and binding to ribosomes

Unlike phenylalanyl-tRNA synthetase from lower eukaryotes, the corresponding enzyme from higher eukaryotes displays a pronounced tendency to associate with ribosomes in vitro. To attempt to uncover the structural features responsible for this difference in behavior, a comparative study of the enzymes purified to homogeneity from sheep liver and yeast was undertaken. The two alpha 2 beta 2-type enzymes displayed remarkably similar subunit molecular masses (71 and 63 kDa for sheep, 74 and 63 kDa for yeast), yet differed markedly in their isoelectric points (8.0 and 5.6 pH units, respectively). Mild tryptic digestion of the enzyme from sheep led to preferential degradation of the 63-kDa beta subunit into two major fragments of 35 and 25 kDa, respectively, with concomitant loss of activity. The isoelectric points of the denatured fragments were found to be distinctly lower than that of the denatured beta subunit, implying that the residues responsible for the basic net charge of the original beta subunit are mainly clustered in a small portion of the polypeptide chain which was excised during proteolysis. Despite their different isoelectric points, the enzymes from yeast and sheep displayed identical requirements for aminoacylation of tRNA at optimal rates. Moreover, the incidence of variations in pH and ionic strength on the kinetic parameters of the two enzymes was indistinguishable. Interpreted in terms of the polyelectrolyte theory, these results support the view that the residues responsible for the basic net charge of the mammalian enzyme are located in a region distal from the active site. It is suggested that the cationic charge of the enzyme allows anchorage to a cellular component carrying negative charges, possibly the ribosome.