The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it]

Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.     

Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.     

Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.     

The citrate-stimulated starch synthase of starchy maize kernels: Purification and properties

Chromatography of maize kernel extracts on DEAE-cellulose resolves two fractions of starch synthase activity, one of which (starch synthase 1) is capable of synthesizing α-glucan in the absence of exogenous primer and the presence of 0.5 m citrate (J. L. Ozbun, J. S. Hawker, and J. Preiss, Plant Physiol. (1971) 48, 765–769). This starch synthase has been purified 200-fold from developing kernels of normal maize, and shown to have no detectable activities of branching enzyme, amylase, pullulanase, phosphorylase, and D enzyme. The preparation, however, was not electrophoretically homogeneous. This preparation had a K_m value of 0.033 mm for ADPglucose in the presence of 0.5 m citrate. The reaction in the presence of citrate was stimulated 10-fold by the addition of excess purified branching enzyme. This stimulation is higher than those reported previously (C. D. Boyer and J. Preiss, Plant Physiol. (1979) 64, 1039–1042) but is consistent with the predicted effects of removal of amylase activity. The effects of salts other than citrate on activity in the absence of exogenous primer were small, but the stimulation could be restored by the addition of excess purified branching enzyme. Citrate increased the affinity of the enzyme for the endogenous primer present to such a level that no effect of exogenous primer on reaction rate could be observed in the presence of 0.5 m citrate. Analysis of the glucan/iodine complex and the enzymatic breakdown products patterns from the products of the starch synthase reaction indicates a high degree of linearity. The results obtained are discussed in relation to the biosynthesis of starch in vivo.     

Catalytic activity of the nucleic acid component of the 1,4-alpha-glucan branching enzyme from rabbit muscles.

2.5 S RNA, the nucleic acid component of the 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase from rabbit muscles, devoid of any protein, catalyses the branching reaction, as does the holoenzyme. The conclusion is drawn that 2.5 S RNA is a ribozyme. To get an insight into the significance of different parts of the molecule for the catalytic activity of 2.5 S RNA, a large fragment isolated from its partial RNAase A digest was investigated. This fragment which proved to be the middle part of polyribonucleotide chain containing all modified nucleotides exerts some catalytic activity, too.     

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.     

植物支链淀粉合成的关键酶——淀粉分支酶

支链淀粉是植物淀粉的主要成分,而淀粉分支酶是其合成的关键酶。淀粉分支酶可分为两同形体家族,本文从酶学特性、染色体定位、基因及基因表达方面阐明了它们之间的联系和区别,并证实不同同形体在植物支链淀粉合成和结构决定上所起作用不同。开展对该酶的深入研究不论是在基础理论研究领域还是在现实应用方面都具重要意义。     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.     

淀粉分支酶3存在于水稻胚乳的淀粉粒之中

对水稻胚乳淀粉颗粒结合的淀粉分支酶进行了研究.酶活性分析表明水稻胚乳中存在着与淀粉颗粒结合的淀粉分支酶.氨基酸测序分析结果表明结合于水稻胚乳淀粉粒的淀粉分支酶是分子量为84 kD的淀粉分支酶3(rice starch branching enzyme 3; RBE3).从开花后5 d到种子成熟,淀粉颗粒结合的RBE3蛋白都保持较为稳定的含量.Northern 分析表明水稻胚乳发育过程中RBE4最先表达而RBE3和RBE1的表达滞后.综合以上研究结果说明RBE3存在于水稻胚乳的淀粉之中是由于RBE3与淀粉葡聚糖链具有较高亲和性而难以和葡聚糖链解离,进而随着淀粉粒的增长而被其包裹.     

Glycogen synthase kinase-2 and phosphorylase kinase are the same enzyme.

The primary structure of the nucleic acid from the branching enzyme 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase (2.5-S RNA) isolated from rabbit muscles has been elucidated. The polyribonucleotide consists of 31 nucleotides; the unique features of the polyribonucleotide are the unusually high content of modified nucleotides (32%) and guanine residues (40%). Apparently 2.5-S RNA belongs to a class of nucleic acids unknown up to now. It is the first time that the structure of a nucleic acid component from a ribonucleoenzyme has been defined. This work is a preprequisite for gaining insight into the intimate activating effect of the poly-ribonucleotide on the enzyme action.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

RNAi沉默淀粉分支酶基因SBEI对玉米直链淀粉合成的影响

淀粉分支酶(SBE)是淀粉合成的限速酶。为了研究SBEI沉默对直链淀粉合成的影响, 克隆了玉米(Zea mays)淀粉分支酶SBEI基因片段, 构建了SBEI的RNAi表达载体pBAC418, 用基因枪将其导入玉米自交系幼胚愈伤组织, 经木糖筛选获得了7株转化再生植株。利用FAD2 intron和xylA基因探针对T₀代再生玉米植株进行DNA dot blot和PCR-Southern检测, 证实5株为阳性植株, 其中4株正常结实。SBEI基因沉默对阳性再生玉米株系籽粒的含油量没有显著影响; 蛋白质含量显著高于受体对照; 总淀粉含量与对照相比无显著差异, 转基因株系直链淀粉含量平均提高了9.8%。     

一株耐热脂肪酶产生菌的筛选及酶学性质研究

从云南省富油地采取了60份土样中,利用透明圈法筛选出一株耐热脂肪酶产生菌。对其酶学性质和发酵条件进行了研究,酶学性质表明,该酶最适作用温度为50℃,最适pH6.0,在pH3.0-8.0范围内稳定,在60℃保温60 min酶活还保留70%;70℃保温60 min残余50%;具有良好的热稳定性;不同金属离子有不同的作用,Ca+,K+对酶有激活作用,Fe3+、Pb2+、Mn2、Cu2+、Al3+、Zn2+对酶活有抑制作用。EDTA对酶影响不大。产酶最佳条件为:MgSO4.7H2O 0.05 g,K2HPO40.1 g,CaCO30.25 g,可溶性淀粉2.5 g,大豆粉2.5 g,装液量50 mL。这株细菌通过培养基优化酶活达到20.3 U/mL。     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Heterologous expression and efficient ethanol production of a <Emphasis Type="Italic">Rhizopus</Emphasis> glucoamylase gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glucoamylases are inverting exo-acting starch hydrolases releasing β-glucose from the non-reducing ends of starch and related substrates. Due to the absence of glucoamylase in Saccharomyces cerevisiae, it is not capable of utilizing starch directly as energy sources without enzymatic or chemical hydrolysis for its ethanol production. In this study, we heterologously expressed a previously isolated Rhizopus arrhizus glucoamylase gene in S. cerevisiae host. The expressed glucoamylase enzyme was secreted into the culture supernatant and exhibited a molecular weight of 68 kDa on SDS-PAGE gel and western blot. In the flask ferment experiment of S. cerevisiae growing on raw starch, the RaGA transformed strains could utilize starch as energy source to produce ethanol up to a final concentration as 5%.