Primary structure and differential expression of beta-amylase in normal and mutant barleys

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Ris? mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.     

Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.     

Multiple molecular forms of β-amylase in seeds and vegetative tissues of barley

The molecular forms of -amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (M_rs) could be recognised. The major isoform present in the mature grain, called isoform B, had an M_r of about 60 000. This was converted on malting or germination to two lower-M_r forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487–491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same M_r as D and a minor low-M_r isoform (E) were present in young developing whole caryopses (8–12 d after anthesis), but not in older developing endosperms (14–21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are green -amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same M_r as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-M_r form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of -amylase are discussed in relation to the recent report that -amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13–16).Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Localization to chromosomes of structural genes for the major protease inhibitors of barley grains

Summary Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous -amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3.     

Serpins in plants and green algae

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.     

Purification and Properties of Three Pig Erythrocyte Carronic Anhydrases

Three distinct forms of the zinc containing enzyme carbonic anhydrase were isolated from pig erythrocytes. One low activity type enzyme and two genetic variants of the high activity type enzyme with identical CO2 hydratase activities which were 8 times as high were isolated from Danish Black and White Swine. In the isolation procedure described, the hemoglobin was eliminated by precipitation with chloroform-ethanol, and the isoenzymes were separated by DEAE-Sephadex chromatography. A number of enzymatically active minor components were separated. They were apparently all genetically linked to one of the three major components. The three purified isoenzymes behaved as homogeneous components during isoelectric focusing and electrophoresis at different pH values. They were characterized in terms of molecular weight, isoelectric pH, zinc content, amino acid composition, and enzymatic activity against CO2, p-nitrophenyl acetate, and β-napthyl acetate. The circular dichroism of the enzymes in the ultraviolet region was studied. The properties of the enzymes were similar to those of carbonic anhydrases of corresponding types isolated from other mammalian species. Sulphur containing amino acid residues were absent in the low activity type enzyme. The amino acid composition of the two high activity mutants deviated only in that an arginine residue in the most widespread genetic variant was replaced by a histidine residue in the less frequent variant. Otherwise the two mutants showed identical properties.     

A recombinant wheat serpin with inhibitory activity

A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27–33% identical with human serpins such as ₁-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with -chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.     

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α-amylase/serine protease inhibitor which inhibits germination-specific cereal α-amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M_r ≈20500 and pI ≈7.2. The amino acid composition and N-teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.     

Isoelectric focusing of subtilisin inhibitors: Detection and partial characterization of cereal inhibitors of chymotrypsin and microbial proteases

Inhibitors of subtilisin and other microbial serine proteases were detected after isoelectric focusing of cereal extracts in polyacrylamide gels by a negative staining technique based on the chromogenic substrate acetyl-d,l-phenylalanine-2-naphthyl ester. The 10 chymotrypsin isoinhibitors identified in experiments with barley extracts were also inhibitors of subtilisin, and sensitivity of detection was about 25 times higher when gels were stained for inhibitors of this enzyme. In addition, one specific subtilisin inhibitor zone was detected. These 11 barley subtilisin isoinhibitors and similar inhibitors in wheat, triticale, rye, oats, sorghum, rice, and maize were further characterized and classified by isoelectric focusing. Techniques used for this purpose included application of microbial enzymes with different specificity of inhibition, comparison of cereal varieties, pretreatment of extract with monospecific antibodies toward a purified inhibitor from the same or a related species, and pretreatment of extract with inhibitor affinity gel. The procedure used for detection of subtilisin inhibitors was also effective after separation of isoinhibitors by sodium dodecyl sulfate-gel electrophoresis.