Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Immunofluorescence localization of α-amylase in the scutellum,germ aleurone and “normal” aleurone of germinated barley grains

Summary Using two step labeling with rhodamine-labeled secondary antibodies, -amylase (EC 3.2.1.1.) was detected in the scutellum, germ aleurone (monolayer partially encircling the scutellum) and normal aleurone (trilayer partially encircling the starchy endosperm; see Fulcher et al. 1972) in sections of Lowicryl-embedded barley (Hordeum vulgare L. cv. Himalaya) after imbibition of whole grains for 24, 48, and 72 h, but not after 2 h. Staining occurred over the protoplasts, cell walls or intercellular spaces of each tissue indicating that all three tissues had secreted as well as produced -amylase. The immunofluorescence in the scutellum was predominantly in the epithelium. Normal aleurone near the aleurone/ scutellum junction showed structural changes indicative of secretory activity by 24 h, and the pattern of cell erosion in the sub-aleurone and starchy endosperm at this and later stages supported this conclusion. The data show that normal aleurone is a major source of -amylase even at early stages of germination, but there is clear evidence also of production and secretion of some -amylase by both the scutellum and the germ aleurone, indicating that these tissues could also contribute to starch hydrolysis.Abbrevations GA₃ gibberellic acid - SDS sodium dodecyl sulphate - TBS 10 mM Tris, pH 7.4, containing 0.15 M NaCl - Tris 2-amino-2-(hydroxymethyl)-1, 3-propanediol     

Repression of α-Amylase Activity by Anoxia in Grains of Barley is Independent of Ethanol Toxicity or Action of Abscisic Acid

Abstract: We studied the effects of anoxia on α-amylase induction, comparing rice ( Oryza sativa L.) and barley ( Hordeum vulgare L.) grains. While gibberellic acid (GA₃) induces α-amylase in rice half-grains under either aerobic or anaerobic conditions, barley half-grains are insensitive to this hormone when applied under anoxia. The possible repressive role of ethanol and abscisic acid (ABA) was investigated. Exogenously added ethanol at concentrations mirroring those found in anaerobically treated tissues was unable to repress α-amylase. The level of ABA in anoxic tissues was found to be much lower than the threshold for α-amylase repression. Overall, the results indicated that these two compounds cannot be held responsible for the failure of barley grains to respond to gibberellic acid. Furthermore, anoxia repressed the induction of α-amylase downstream of the slender mutation, indicating that the repression is independent of effects related to gibberellin perception. Overall, the results suggested that the ability of rice to respond to gibberellins under anoxia is an adaptative trait, independent of known negative regulators of α-amylase induction.     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

The presence of two types of β-cyanoalanine synthase in germinating seeds and their responses to ethylene

Germinating seeds of many species contain two types of β-cyanoalanine synthase (CAS, EC 4.4.1.9) that convert HCN to β-cyanoalanine. One is cytoplasmic CAS (cyt-CAS), which is precipitated by 50 to 60% (NH₄)₂SO₄ and has a pH optimum of 10.5. Cytoplasmic CAS is present at high levels in dry seed and its activity does not increase during imbibition. The activity of cyt-CAS is not affected by exogenously applied ethylene (C₂H₄), except in rice ( Oryza sativa cv. Sasanishiki). The second type of CAS found in seed is mitochondrial CAS (mit-CAS), which is precipitated by 60 to 70% (NH₄)₂SO₄ and has a pH optimum of 9.5. Mitochondrial CAS is present at low levels in dry seed, and its activity increases greatly during imbibition in the seeds of all species tested. Exposure to C₂H₄ stimulated mit-CAS activity in seeds of rice, barley ( Hordeum vulgare cv. Hadakamugi). cucumber ( Cucumis sativus cv. Kagafushinari) and cocklebur ( Xanthium pennsylvanicum ). The increase in the mit-CAS activity in cocklebur in response to C₂H₄ commenced alter a lag period of 2 to 3 h when the duration of soaking was short (16 h), but commenced without a lag period when the seeds were soaked for three months. Application of both chloramphenicol and cycloheximide to the axial and cotyledonary tissues of cocklebur seeds strongly inhibited growth as well as the increase in mit-CAS activity. It is postulated that the mit-CAS is synthesized de novo during imbibition and that its activity is regulated by C₂H₄, CO₂ which also promotes seed germination in some species, was ineffective m stimulating mit-CAS activity in cocklebur seeds.     

Differential expression of α-amylase genes in germinating rice and barley seeds

Steady-state levels of mRNA from individual -amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the -amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, -amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenver two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley -amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI -amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI -amylases always preceded those encoding low-pI -amylases. Two distinct differences in -amylase gene expression were observed between the two barley varieties. levels of high-pI -amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI -amylase mRNA and nearly four times as much low-pI -amylase mRNA than the slower-germinating Himalaya variety.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Differential Proteolysis of the Full-Length Form of the L-Type Calcium Channel α1 Subunit by Calpain

Abstract: This study examines the proteolysis of the carboxy terminal domain of the full-length (α1₂₁₂) and truncated (α1₁₉₀) forms of the rabbit skeletal muscle L-type calcium channel α1 subunit by calpain I and calpain II. Although both forms of the α1 subunit show little sensitivity to proteolysis by calpain II, α1₂₁₂ is relatively more sensitive than α1₁₉₀ to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of α1₂₁₂ in a time- and concentration-dependent manner and proteolysis is independent of the α1₂₁₂ phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from α1₂₁₂ and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Composition of α-amylase secreted by aleurone layers of grains of himalaya barley

α-Amylases secreted by the aleurone layer of whole barley grains were relatively rich in histidine and relatively poor in glutamate/glutamine and serine when compared to other eukaryotic proteins. The secreted α-amylases had an estimated 0.5 residues each of glucose, mannose and N-acetylglucosamine per molecule of protein (MW 41 400 daltons), and gave positive staining reactions for carbohydrate on sodium dodecylsulfate polyacrylamide gels. Because the average α-amylase molecule had less than one sugar residue per enzyme molecule, it was concluded that secreted α-amylases were heterogeneous with respect to glycosylation. A second protein co-purified with α-amylase, but the amino acid composition of this protein was different from that of barley or wheat α-amylase. This protein was composed of two 21 500 dalton polypeptides. No significant amounts of L-leucine (¹⁴C-U) were incorporated into this second protein in isolated aleurone tissue during incubation with gibberellic acid, perhaps because much of it was already present in the starchy endosperm at the time of hormone addition.     

Amylase from Lactobacillus cellobiosus D-39 isolated from vegetable wastes: Purification and characterization

An organism producing α-amylase and identified as Lactobacillus cellobiosus D-39 was recently isolated from vegetable wastes. Its amylase was purified by (NH₄)₂SO₄ precipitation and DEAE cellulose column chromatography and was obtained in crystalline form. It was fairly stable at a broadly neutral pH range and maximally active at 50°C. The molecular weight was 22 500 daltons as determined by SDS gel electrophoresis.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.