Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers

The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being α-amylase. The mRNA for α-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.     

Synthesis of a possible precursor of alpha-amylase in wheat aleurone cells

α-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific λ-globulin for α-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic α-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of α-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of α-amylase in aleurone cells.     

Biosynthesis of alpha-Amylase in Vigna mungo Cotyledon

In vitro translation of RNA extracted from Vigna mungo cotyledons showed that α-amylase is synthesized as a polypeptide with a molecular mass of 45,000, while cotyledons contain a form of α-amylase with a molecular mass of 43,000. To find out whether the 45,000 molecular mass polypeptide is a precursor to the 43,000 found in vivo, the cell free translation systems were supplemented with canine microsomal membrane; when mRNA was translated in the wheat germ system supplemented with canine microsomes, the 45,000 molecular mass form was not processed to a smaller form but the precursor form was partly processed in the membrane-supplemented reticulocyte lysate system. When V. mungo RNA was translated in Xenopus oocyte system, only the smaller form (molecular mass 43,000) was detected. Involvement of contranslational glycosylation in the maturating process of the α-amylase was ruled out because there was no effect of tunicamycin, and the polypeptide was resistant to endo-β-H or endo-β-D digestion. We interpret these results to mean that the 45,000 molecular mass form is a precursor with a signal peptide or transit sequence, and that the 43,000 molecular mass is the mature form of the protein.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [³⁵S]methionine into the α-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-α-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify α-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that α-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of α-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of α-amylase.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.

     

Developmental Differences in Embryos of High and Low Protein Wheat Seeds during Germination

Developmental patterns of embryos from high and low protein wheat (Triticum aestivum) grain produced under varied fertilizer conditions were compared. High protein grain produced seedlings 25% heavier with 25% more total RNA, 30% more DNA, 40% more amino acids, 60% more ribosomes, and 80% more soluble protein content than that of low protein seed. Consistently higher glutamine synthetase and α-amylase and lower acid phosphatase activities were observed in high protein seeds, though the isozyme pattern of α-amylase was not different in the two kinds of seeds. The high total ribosomes and particularly, polysome content observed in high protein seeds may be responsible for the rapid growth and high yield of these seeds.     

An endogenous alpha-amylase inhibitor in barley kernels

Barley (Hordeum distichum cv Klages) kernels were shown to contain a factor that converted malted barley α-amylase II to the α-amylase III form. After purification by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gel-filtration on Bio Gel P60, the factor gave a single band of protein on isoelectric focusing. The purified factor inhibited hydrolysis of soluble starch by α-amylase II from malted barley and germinated wheat (Triticum aestivum cv Neepawa). However, α-amylase I from these cereals was not affected. The inhibitor was not dialyzable and was retained by a PM 10 ultrafiltration membrane suggesting a molecular weight greater than 10,000 daltons. Heat treatment of the inhibitor at 70°C for 15 minutes at pH 5.5 and 8.0 resulted in considerable loss of inhibitory activity.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.     

Alpha-amylase synthesis in wheat kernels as influenced by the seed coat

The effect of seed coat removal on the synthesis of α-amylase isoenzymes in wheat was investigated. The immature wheat endosperm-aleurone (seed coat and embryo detached) produced considerably less α-amylase activity than immature whole or de-embryonated wheat kernels, when incubated under identical conditions of 18.5 C and 99% humidity, in the presence or absence of gibberellic acid (GA₃). The incubated endosperm-aleurone also exhibited unique α-amylase isoenzyme composition when compared to the isoenzyme compositions of incubated whole and de-embryonated immature and mature wheat kernels both in the presence or absence of GA₃. Subsequent studies indicated that the seed coat may contain factor(s) required for normal α-amylase isoenzyme synthesis.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable α-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

Water stress enhances expression of an alpha-amylase gene in barley leaves

The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were β-amylase and the other, α-amylase. The α-amylase had the same isoelectric point as one of the gibberellin-induced α-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone α-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.

In unwatered seedlings, leaf α-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on β-amylase. α-Amylase occurred uniformly along the length of the leaf but β-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf α-amylase occurred inside chloroplasts.

Leaf radiolabeling experiments followed by extraction of α-amylase by affinity chromatography and immunoprecipitation showed that increase of α-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled α-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more α-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes.

     

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

The most abundant α-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This α-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an α-amylase by polarimetry, substrate specificity, and end product analyses. The purified α-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This α-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. β-glucan) are not substrates of the enzyme. A very low amount of this α-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or α-amylase which is integrally associated with the chloroplast.     

Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.     

Embryoless Wheat Grain: A Natural System for the Study of Gibberellin-induced Enzyme Formation

Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support α-amylase synthesis only in the presence of gibberellin A₃ (GA₃). In the absence of GA₃ some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA₃ induction of α-amylase. The level of β-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA₃, β-amylase decreases at a slower rate. Isoenzymes of α-amylase from GA₃-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 μg/ml) completely inhibits the synthesis of α-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 μg/ml) was ineffective while 6-methylpurine (60 μg/ml) gave 65% inhibition without decreasing the number of isoenzymes.