Glycosylation of human α1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Human α₁-antitrypsin (α₁-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α₁-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α₁-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α₁-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α₁-AT. The human α₁-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α₁-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α₁-AT in S. cerevisiae. The α₁-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α₁-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.     

Per-O-methylated α- and β-CD: Cyclodextrins with Inverse Hydrophobicity

The investigation focuses on the computer-aided generation of the molecular geometries, contact surfaces, and lipophilicity patterns of per-O-methylated α-CD ( 1 ) and its β-CD homolog 2 , and compares them with their parent non-substituted cyclodextrins. The molecular geometries, compared via statistical analysis of crystal structure data available, reveal 1 and 2 to be considerably more flexible than α-and β-CD, allowing wide variations in the tilting of the glucose units relative to the macrocyclic ring axes. The comparative evaluation of their contact surfaces not only discloses a substantial increase of the torus heights upon per-O-methylation (from ∼8.0Å in α- and β-CD, to → 11.1Å in 1 and 2 ), but also an enlargement of their cavity areas by 40% (+35Ų for α-CD → 1 ) and 70% (+75Ų for β-CD → 2 ), respectively. The hydrophobic characteristics of 1 and 2 , emerging from the molecular lipophilicity patterns (MLPs) generated and projected onto the contact surfaces in color-coded from, are inverse to those for α- and β-CD: the most hydrophobic surface regions of 1 and 2 are located at the torus rims made up by the 2-OMe and 3-OMe groups at one side, and the 6-CH₂OMe moieties at the other, with a hydrophobic „band”︁ wrapping around the outside of the macrocycles; these „exo-lipophilic”︁ topographies are opposed by pronouncedly hydrophilic central cavities. A variety of experimental findings can be rationalized on the basis of the opposite lipophilicity profiles of the CDs and their permethylated analogs, such as for example the opposite orientation of benzaldehyde, p-nitrophenol, and 3-iodopropionic acid in the cavities of α-CD and of 1 . Thus, the notion is substantiated that the operation of dispersive interactions between guest and CD-host cavities play a more dominant role in inclusion complex formation than hitherto appreciated.     

Purification and Characterization of a Maltotriogenic α-Amylase I and a Maltogenic α-Amylase II Capable of Cleaving α-1,6-Bonds in Amylopectin

Extracellular α-amylases I and II, produced by a facultative thermophile Bacillus thermoamyloliquefaciens KP 1071 capable of growing at 30–66°C, were purified to homogeneity. α-Amylase I consisted of a single polypeptide with methionine residue at the NH₂-terminus. α-Amylase II consisted of two equivalent polypeptides each comprising a methionine at the NH₂-terminus. α-Amylase I hydrolyzed endotypically α-1,4-bonds in glycogen, amylopectin and β-limit dextrin, but not their α-1,6-bonds. α-Amylase II degraded amylopectin and β-limit dextrin in exo-fashion by cleaving preferentially α-maltose units from the non-reducing ends and hydrolyzing their α-1,6-branch points. α-Amylase II hydrolyzed maltotriose, phenyl-α-maltoside, α- and β-cyclodextrins and pullulan, whereas α-amylase I had no activity for all these sugars. α-Amylases I and II hydrolyzed maltotetraose, maltopentaose, α-limit dextrin and amylose, but they were inactive for maltose, isomaltose and panose. It was suggested that α-amylase I is the most thermostable type of hitherto known maltotriogenic endo-acting α-amylases, and α-amylase II is the first maltogenic exo-acting α-amylase able to split α-1,6-bonds in amylopectin.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

α-GLUCOSIDASE, α-GLUCOSIDE PERMEASE,MALTOSE FERMENTATION AND LEAVENING ABILITY OF BAKER'S YEAST

α-Glucosidase and α-glucoside permease are synthesized simultaneously in two different strains of baker's yeast (Saccharomyces cerevisiae) when the cells are induced with maltose. α-Thioethyl D-glucopyranoside (α-TEG) inhibits the transport of maltose into the cells, and the transport of maltose and α-TEG appeared to be mediated by the same permease system. There is an obvious correlation between α-glucosidase, α-glucoside permease and maltose fermentation activities in the yeasts while no correlation between these and the leavening ability of the yeasts can be demonstrated. Apparently the glucose concentration in dough is high enough to inhibit the permease-mediated transport of maltose into the cells thus impairing leavening ability from the maltose fermenting system.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

Tannin fraction from Ampelopsis grossedentata leaves tea (Tengcha) as an antioxidant and α‐glucosidase inhibitory nutraceutical

Leaf of Ampelopsis grossedentata is a new resource of functional foods with healthful properties. Antioxidant and α‐glucosidase inhibitory activities of water extract (made in the style of drinking), tannin fraction (TF) and dihydromyricetin (DMY) from A. grossedentata leaves were evaluated. The main component of TF was identified as gallotannins. DPPH and ABTS radical scavenging activities and reducing power of TF were superior to those of water extract, however, inferior to those of DMY. In no PBS wash protocol of cellular antioxidant activity assay, DMY and TF exhibited similarly, while in PBS wash protocol, the value of TF was higher than that of DMY. In addition, TF possessed the highest α‐glucosidase inhibitory activities (IC₅₀ = 1.94 μg mL^?1), followed by water extract (IC₅₀ = 23.10 μg mL^?1) and DMY (IC₅₀ = 72.21 μg mL^?1). The strong α‐glucosidase inhibitory activity of TF may attribute to the binding capacity to enzymes, as confirmed by fluorescence analysis.     

The Effect of Substrate Diffusion Factor on Immobilized α-Amylase

α-Amylase of Bacillus subtilis was immobilized on acrolein-styren copolymers of spongy and non-spongy structures which had been modified with hexamethylenediamine followed by glutaraldehyde. Optimum bonding rates were determined from the immobilization experiments carried out with different amounts of α-amylase, pH-activity relationships, variation of reaction rates with the time due to the substrate diffusion limitation factor and the observed K_m values were evaluated.     

Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

Coupling Reaction of Bacillus macerans Cyclodextrin Glucanotransferase on Glucosyl-α-cyclodextrin and Glucose

Treatment of glucosyl-α-cyclodextrin with radioactive glucose in the presence of Bacillus macerans cyclodextrin glucanotransferase, yielded radioactive maltose, maltotriose, and four kinds of glucosyl branched oligosaccharides. The branched oligosaccharides formed had identical Rf values as those of branched oligosaccharides of 5-8 glucose unit on paper chromatography using the solvent system of 1-butanol: pyridine: water (6/4/4), (Fractions B₅-B₈). Individual oligosaccharides were isolated and their structures determined using the action of porcine pancreatic α-amylase, β-amylase, pullulanase, isopullulanase and glucoamylase. The structure of the main component of the fraction B₅ was 6⁴-O-α-glucosylmaltotetraose, B₆ was 6⁴-O-α-glucosylmatopentaose, B₇ was 6⁵-O-α-glucosylmaltohexaose, and B₈ was 6⁶-O-α-glucosylmaltoheptaose. Fraction B₈, which formed at the initial stage of the reaction, contained 6⁴, 6⁵ and 6⁶-O-α-glucosylmaltoheptaose. Reducing end ¹⁴C-labelled 6⁴-O-α-glucosylmaltotetraose was degraded to 6³-O-α-glucosylmaltotriose and radioactive glucose by the exhaustive action of Bacillus macerans cycloamylose glucanotransferase, and 6³-O-α-glucosylmaltotriose was completely non-reactive to the enzyme. From these results, we proposed a model of the active site of the enzyme.     

Characterization of an Exo-Maltotetraose-forming Amylase of Pseudomonas stutzeri and Application in p-Nitrophenyl β-D-Galactosyl-α-Maltooligosaccharides Production

Some kinetic characteristics and hydrolytic action patterns on various β-D -galactosyl-maltooligosaccharides (Gal-Gn), ranging in size from D.P. (degree of polymerization) 5 to 8, of an exo-maltotetraose-forming amylase of Pseudomonas stutzeri (G4-amylase) were examined to produce a few p-nitrophenyl β-D -galactosyl-α-maltooligosaccharides (Gal-GnP, n = 4,5). The relative hydrolytic reaction rates for larger Gal-Gn by the enzyme were larger than those for smaller saccharides tough the values for unmodified linear maltooligosachharides were almost same. Michaelis constants (Km) for hydrolysis of Gal-G4, Gal-G5, Gal-G6 and Gal-G7 by the enzyme were 1.3, 1.9, 1.3 and 1.3mM, and apparent molecular activities (ko) for these saccharides were 5.9, 38, 91 and 126s⁻¹, respectively. The values of ko/Km for them were remarkably smaller than those for unmodified linear maltooligosaccharides. The G4-amylase cleaved 2 points of the α-1,4-glucosidic linkage in β-1,4-Gal-G4 to give β-1,4-Gal-G2 and -Gal-G3 in the molar ratio of 3:1, whereas the enzyme attacked 3 points of the linkage in β-1,4-Gal-G5, -Gal-G6 and -Gal-G7 to form β-1,4-Gal-G2,-Gal-G3 and Gal-G4 in the molar ratios of 2:5:1, 1:3:6 and 1:3:6, in the early stage of the reaction, respectively. On the other hand, the enzyme showed no action on β-1,6-Gal-G4 and formed β-1,6-Gal-G4 solely from β-1,6-Gal-G5, and β-1,6-Gal-G4 and -Gal-G5 were from β-1,6-Gal-G6 and -Gal-G7 in the ratios of 8:1 and 2:1, respectively. The enzyme also catalyzed the transfer action to produce Gal-G3P, Gal-G4P and Gal-G5P, of which the formation ratio was coincided well with the hydrolytic action pattern on each Gal-Gn, from Gal-Gn tested as a donor and p-nitrophenyl α-glucoside (GP) as an acceptor in an aqueous solution containing 40% (v/v) methanol. By using this novel reaction, Gal-G5P is now producing on an industrial scale to apply as a substrate for the assay of human α-amylase.     

Effect of β‐cyclodextrin on pasting properties of wheat starch

Changes of viscosity characteristics of genetically diverse hexaploid wheat starches during pasting in water, 1% NaCl solution, or at pH 4 or pH 10 were studied using a Rapid Visco‐Analyzer. Peak viscosity (PV( hot paste viscosity (HPV) and cool paste viscosity (CPV) of all the wheat starches was little affected in pH 4 and pH 10 treatments. In 1% NaCl, all starches showed substantial increases in all three parameters relative to pasting in water. The use of 1% β‐cyclodextrin (β‐CD) (cycloheptaamylose) solution increased PV of high‐swelling starches, but generally slightly decreased that of low swelling starches in all treatment conditions. HPV was always reduced by addition of 1% β‐CD, but CPV was increased in most treatments for Anza and Yecora Rojo (low swelling( but decreased for Klasic (high swelling). Bacterial α‐amylase was added to starch or flour. The effect of β‐CD was shown to be independent of α‐amylase inhibition in wheat starch, but β‐CD strongly inhibited α‐amylase in wheat flour.     

Comparative behaviour of goat β and αs1-caseins at the air–water interface and in solution

Here we present a comparative study of caprine β- and α_s1-caseins behaviours at the air–water interface and in solution. Both caseins were purified from the milk of a single goat homozygous at the α_s1- and β-Cn loci, with a high degree of purity (98%). Physical measurements (ellipsometry, surface pressure and surface rheology) were performed at the air–water interface, whereas SAXS measurements were performed on casein solutions. Our results clearly show that self-organizations, both at the air–water interface and in solution are different for β- and α_s1-caseins. β-casein is unfolded in solution and forms a network at the interface, while α_s1-casein forms compact objects in solution and is organised in fluid domains at the interface. We also show that the presence of Ca²⁺ in the subphase strongly disturbs the interfacial layer formed by the caseins. It is elsewhere worth noting that in solution, the aggregation of α_s1-casein induced by calcium ions is associated with a pronounced change in the molecular structural organisation of the protein, which seems to adopt, in these conditions, an unfolded structure.     

Chemical Constituents of Gold‐red Apple and Their α‐Glucosidase Inhibitory Activities

Ten compounds were isolated and purified from the peels of gold‐red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β‐dihydroxyursan‐28‐oic acid (1), 2α‐hydroxyoleanolic acid (2), euscaphic acid (3), 3‐O‐p‐coumaroyl tormentic acid (4), ursolic acid (5), 2α‐hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α‐linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α‐Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α‐glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC₅₀ = 15.19 μM). Ultra‐performance liquid chromatography‐electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes.     

α-D-Polyglucane-Iodine Complexes

The temperature-dependent dyeability of oligoglucanes and polyglucanes with I₂/KI solution is based on the interaction between iodine and the C-atoms of glucosidic bonds (Vetter and Thorn [1, 2]). Only when the helix is compressed, the expansion of the lumen that is necessary for the attachment of iodine bands is achieved as suggested by Freudenberg et al. in 1939 [3]. The development of colour starts with the maltododecaosis (just over two spirals) within a temperature range of 20 to 25°C adsorbing one pentaiodide and bringing about a pale shade of pink. The rich blue tone is achieved with pure amyloses forming a maximum of about 610 to 620 nm. Unramified polysaccharides which had been synthesised by our team from 30 glucoside residues, still showed a purple colour with a maximum of 510 nm at temperatures from 20 to 25°C. Defined polysaccharides from 40 residues are bluish-purple (just 7 spirals of a tension-free helix) with a maximum absorption of about 550 nm. According to our knowledge, the rich blue tone requires at least 50 glucoside residues linked in an unramified chain [1, 2]. Amylopectin from potatoes has a maximum of 575 to 580 nm which shifts to 555 to 560 nm after partial break-down into β-dextrin by β-amylase (EC 3.2.1.2) and does a long-wave re-shift to 570 nm after splitting off of the side-chain stumps in the basic structure by pullulanase (EC 3.2.1.41). In the case of maize, the maximum is 535 nm for amylopectin, 520 nm for β-dextrin, and 555 nm for the basic structure. This means that in amylopectin from potatoes the helical sections available for the adsorption of iodine bands are longer than those in amylopectin from maize [4]. In glycogen iodine accumulates in a diffuse manner without forming any long bands. For this reason, an absorption shoulder of merely 400 to 500 nm is found by photometry [4]. A treatment with β-amylase makes the situation even worse; only when the side-chain stumps are separated by pullulanase does the formation of iodine bands in the basic structure of the glycogen improve to a maximum of 500 nm. This maximum corresponds to unramified sections of less than 30 glucoside residues [4, 2, 6].     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.     

Hydrolysis of Cyclodextrin by Aspergillus oryzae α-Amylase

The hydrolysis of α-, β- and γ-cyclodextrins by Aspergillus oryzae α-amylase was studied at pH 5.2 and 37°C. The kinetic parameters were determined and it was found that the V _max value increased markedly in the order α-, β- and γ-cyclodextrin, but no significant difference was observed in the K_m values. The qualitative and quantitative distribution of the hydrolysis products were determined by HPLC. In the case of γ-cyclodextrin the time course of the kinetic parameters was compared to the qualitative and quantitative distribution of the hydrolysis products.     

Post mortemRelease of Fish White Muscle α-Actinin as a Marker of Disorganisation

α-Actinin release and its degradation from myofibrils Z-line were studied in post mortem white dorsal muscle from bass and sea trout stored at 4°C and 10°C. Using α-actinin specific antibodies, we show that this protein is rapidly released within the first 24 h for the two species, and reaches a plateau within 4 days. Proteolysis take place very rapidly in bass muscle yielding 80 and 40 kDa fragments from α-actinin as major bands of proteolysis. Sea trout muscle is more resistant, and muscle stored at 4°C is not significantly α-actinin degraded even 10 days after death. In the case of sea trout muscle stored at 10°C, an increasing quantity of 80 and 40 kDa fragment can be observed after the third day. These results show that release and proteolysis of α-actinin are time- and temperature-dependent processes that take place at the early stages of fish storage. Furthermore, we observed that proteolysis of α-actinin seems to be dependent on fish species. In both species studied, the early release of α-actinin comes before the degradation of released molecules, and appears as a biphasic process throughout the disorganisation of post mortem muscle in fish cold-stored above 0°C.     

Secretion of Mouse α-Amylase from Kluyveromyces lactis

We constructed two mouse α-amylase secretion vectors for Kluyveromyces lactis using the well-characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α-amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non-glycosylated α-amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four-fold more α-amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins. © 1997 John Wiley & Sons, Ltd.     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.