Purification and differential expression of enolase from maize

Enolase was purified from maze ( Zea mays L. inbred B73)seeds to a 55 and 56 kDa protein doublet based upon sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Purification included ammonium sulfate-precipitation, gel filtration, Mono Q, and Phenyl Superose chromatography. Two-dimensional gels further resolved the 56 kDa protein into three isoselectric forms. Polyclonal antibodies raised against the purified proteins, were found to bind specifically to both the 55 and 56 kDa proteins during purification. Theses antibodies did not recognized a 56 kDa protein when the strain was complemented with maize enolase (pZM245). Maize enolase antibodies recognized a extracts indicated that the 55 kDa form of enolase was more abundant in roots. Enolase protein levels remained unchanged in maize roots after 24 h of anaerobiosis, even though the specific activity of enolase increased to twice its initial levels. A plastid form of enolase in maize could not be found as either enolase activity or protein (with immunoblots).     

A ca. 40 kDa maize (Zea mays L.) embryo dehydrin is encoded by the dhn2 locus on chromosome 9

Dehydrins (LEA D11 proteins) are the products of multigene families in a number of higher plants [5]. To date, however, only one dehydrin locus, dhn1 (a major embryo and drought-induced protein of ca. 18 kDa) has been placed on chromosome 6L of the genetic linkage map of maize. The presence of a larger, ca. 40 kDa embryo protein that is also specifically detected by anti-dehydrin antibodies had been observed in some maize inbreds, including B73, suggesting that other dhn loci may exist. The ca. 22 kDa and ca. 40 kDa immunopositive proteins were purified from B73 and their amino acid compositions determined. The two proteins' amino acid compositions are typical of dehydrins, yet they differ from each other, indicating that they are distinct dhn gene products. Different size alleles for both proteins, or presence/absence in the case of the ca. 40 kDa protein, were evident from comparisons of embryo proteins of various maize inbreds. Analysis of segregating F₂ progeny derived from self-pollination of F₁ hybrids from four crosses (B73 × OH43, Mo17 × A632, AHO × A632, Latente × A632) revealed that alleles of the two genes assort independently. Map positions of the two dhn loci were then determined using two maize recombinant inbred line (RIL) mapping populations. The predicted map position of the gene controlling production of the ca. 22 kDa protein confirmed that this protein is the product of the dhn1 gene. The gene encoding the ca. 40 kDa dehydrin-like protein maps to a new locus on chromosome 9S near wx1, which we have named dhn2.     

Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm,Bombyx mori

A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 × 10⁻⁸ M for JH I, 1.14 × 10⁻⁷ M for JH II and 3.9 × 10⁻⁷ M for JH III, and thus its affinity for JH analogues is in the order of JHI >JHII >JHIII. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.     

Phosphorylation of aortic plasma membranes by protein kinase C

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca²⁺ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca^2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca²⁺.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - K_m Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate     

Purification of a 41 kDa cod-allergenic protein

Cod fish is one of the foods most frequently involved in allergy. Only the cod allergen Gad c I, a 12.3 kDa parvalbumin, has been purified and characterized. Recently, we have detected allergen bands which have not previously been described, in particular a 41 kDa protein, by Western-blot. In the present work, this protein has been purified from a crude cod extract by ammonium sulfate fractionation, hydroxyapatite chromatography and preparative electrophoresis; a single band with an M_r of 41×⁻³ was found in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition and the isoelectric point of the protein were determined. The purified protein (p41) was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals and to a monoclonal anti-parvalbumin which recognizes specifically the first calcium binding site of parvalbumins. p41 may therefore contain a calcium binding site corresponding to an IgE-epitope similar to that of Gad c I.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Tillering in the sugary1 sweet corn is maintained by overriding the teosinte branched1 repressive signal

The evolution of apical dominance in maize during domestication from teosinte is associated with higher expression from the teosinte branched1 (tb1) gene that inhibits tiller bud outgrowth. Unlike many standard maize varieties, the sweet corn inbred P39 that carries a mutation in a starch biosynthesis gene sugary1 produces multiple tillers and providing an opportunity to explore the diversification of the tb1 signal in maize. Through gene expression analysis, we show that tiller buds in P39 continue to grow by overriding the high expression level of tb1 that arrests bud outgrowth in maize inbred B73. In addition, we demonstrate that while B73 is largely non-responsive to shade, both P39 and teosinte respond through tb1-independent and tb1-dependent molecular mechanisms, respectively, leading to inhibition of tiller bud outgrowth.     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

Separation and analysis of the major forms of plasma fibronectin

Human plasma fibronectin exists in circulation in multiple molecular forms that are distinguishable by SDS-polyacrylamide gel electrophoresis (zone I, approx. 450 kDa dimers; zone II, 190-235 kDa; Zone III, 146-175 kDa). (Chen, A.B., Amrani, D.L. and Mosesson, M.W. (1977) Biochim. Biophys. Acta 493, 310-322). We report here on investigations of plasma fibronectin that had been purified from the 'heparin-precipitable fraction' of plasma by DEAE-cellulose chromatography using buffers containing a chaotropic salt (KSCN). Zone I fibronectin and zone II fibronectin were subsequently separated by Sepharose CL-6B chromatography in the presence of 0.3 M KSCN. Electrophoresis of reduced zone I fibronectin dimers showed the presence of three types of subunits (i.e., 220 kDa, 215 kDa, 207 kDa), evidently all having the same NH2-terminal sequence. Subunits of this size were also found in reduced zone II fibronectin, as well as another polypeptide of 190 kDa, the latter amounting to under 5% of the total. Unreduced zone I fibronectin was resolved by gel electrophoresis into a doublet. The upper component amounted to approx. 90% of the total and was comprised of 220 kDa and/or 215 kDa subunits; the lower component contained 207 kDa plus a 220 kDa or 215 kDa subunit. Scanning transmission electron microscopy indicated that under physiologic conditions zone II fibronectin molecules, like those in zone I, exist as pleiomorphic, loosely folded structures (approx. 16 X 8-12 nm) that are somewhat smaller than dimeric zone I molecules (approx. 24 X 16 nm). Circular dichroic spectral analyses suggests that both types have similarly folded local domains. Affinity chromatography experiments revealed a relative decrease in the binding of zone II fibronectin to gelatin but no difference from zone I fibronectin with respect to heparin or fibrin binding.     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

Subsite mapping of purified glucoamylase I, II, III produced by Arthrobotrys amerospora ATCC 34468

Arthrobotrys amerospora ATCC 34468 produced glucoamylase in a medium containing maize starch as carbon source. On native PAGE, crude glucoamylase showed three isoenzymes which were designated as Glu I, Glu II, Glu III according to their electrophoretic mobility. These were purified by column chromatography techniques. The energy of binding for each glucoamylase was calculated using Hiromi's kinetic based calculation. At subsite 1, the binding energies for Glu I, II and III were found to be negative.     

Genotype-specific heat shock proteins in two maize inbreds

Summary Leaf blade tissue of maize inbred lines B73 and Mo17 was analyzed for intraspecific genetic variability in the heat shock response. The maize inbreds were characterized for acquired thermal tolerance and patterns of heat shock protein synthesis. The leakage conductivity assay of membrane stability during stress indicated that Mol7 possesses greater potential than B73 to acquire thermal tolerance. Poly(A)⁺ RNA, extracted from leaf blades, was translated in vitro in the presence of ³⁵S-methionine and the translation products separated by twodimensional gel electrophoresis. Major genotypic differences were observed in the translation products. Mo 17 synthesized twelve unique heat shock proteins in the 15–18 kD range, but B73 synthesized only three unique heat shock proteins in the same range. DNA polymorphisms were observed between the maize lines using ³²P labeled heat shock protein gene probes.Abbreviations HKT Heat-killing time - HS Heat shock - HSP Heat shock protein - HMW High molecular weight - LMW Low molecular weight Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-333     

Carbohydrate and lignin contents of plant materials used in animal feeding

A total of 115 samples representing 38 different feedstuffs was analysed for carbohydrates and lignin. The samples were analysed for low-molecular weight (LMW) sugars by high-performance liquid chromatography, starch, fructan and mixed linked β(1 → 3;1 → 4)-D-glucan by colorimetry, total, soluble and insoluble non-starch polysaccharides (NSP) by gas-liquid chromatography and lignin by gravimetry. For all but alfalfa meal, almost quantitative recovery of carbohydrates and lignin was obtained with a deviation between calculated and analysed values of less than 2 g kg⁻¹ dry matter. The correlation between calculated and analysed values was 0.985 (P < 0.0001).The concentration (g kg⁻¹ dry matter) of LMW-sugars varied from 5 g kg⁻¹ and up to 137 g kg⁻¹ with the lowest values found in cereal substitutes, whole grain cereals and by-products while the protein concentrates in general had the highest content of LMW-sugars (57–137 g kg⁻¹). Starch was the main polysaccharide in whole grain cereals where it varied from 468 g kg⁻¹ in oats to 690 g kg⁻¹ in maize, in cereal by-products (93–902 g kg⁻¹) and in tapioca (768 g kg⁻¹). In contrast, the concentration of starch was low in all protein concentrates but peas and faba beans. The lowest levels of NSP and lignin were found in maize flour (NSP, 21 g kg⁻¹; lignin, 4 g kg⁻¹) and the highest levels in oat hull meal (NSP, 503 g kg⁻¹; lignin, 148 g kg⁻¹). There was also a significant variation in NSP and lignin in protein concentrates with the NSP value varying from 189 g kg⁻¹ in faba beans to 451 g kg⁻¹ in white lupins and with lignin varying from 12 g kg⁻¹ in white lupins to 133 g kg⁻¹ in sunflower cake. Grass meal, alfalfa meal and sugar beet fibre had in general high concentrations of NSP and lignin with values in grass and alfalfa meals of NSP: 329–426 g kg⁻¹ and lignin: 128–169 g kg⁻¹ and in sugar beet fibre 779 g kg⁻¹ and 35 g kg⁻¹, respectively.     

Relationship between activities of key enzymes involved in starch synthesis and accumulation in maize inbred lines during grain filling

Time course of starch production and the key enzyme activities in the grains of four maize inbred lines (two high-starch and two low-starch lines) were studied. Accumulation of grain starch and its components in four maize inbred lines rose continuously after pollination and increased as a sigmoid curve during grain filling. The accumulation rates showed single-peak curves. The accumulation rates of starch and its components reached their peaks on 25–32 days after pollination (DAP), respectively. Activities of adenosine diphosphoglucose pyrophosphorylase (AGPPase) and starch synthase in the grains of four lines followed single-peak curves with the peaks on 24–31 DAP. The highest activity of the starch-branching enzyme (Q-enzyme) in the grains of both high-starch lines appeared on 23 DAP, but that of both low-starch inbred lines showed double-peak curves, the peaks being at 15–20 DAP and 30–35 DAP. There was significant positive correlation between AGPPase, soluble starch synthase (SSS), and starch granule-bound synthase (GBSS) activities. The results indicated the Q-enzyme had different expression patterns in the high-and the low-starch maize inbred lines, and that AGPPase, SSS, and GBSS activities were significantly and positively correlated with amylose, amylopectin, and starch accumulation rates in all lines. This text was submitted by the authors in English     

Overexpression,Purification, and Characterization of the Recombinant Leucine Aminopeptidase II of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His₆-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-p-nitroanilide, followed by Arg- and Lys-derivatives. The His₆-tagged enzyme was stimulated by Co²⁺ ions, but was strongly inhibited by Cu²⁺ and Hg²⁺ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co²⁺ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme. Received: 16 August 2002 / Accepted: 4 September 2002     

Pectin transeliminase complex fromAspergillus flavus

Aspergillus flavus grown in a liquid medium containing pectin as the sole carbon source produced extracellular enzymes which degraded the 1,4-α-d-glycosidic bonds of pectin. The products of degradation were characteristic of substances produced by transeliminase. Synthesis of this enzyme was repressed by the addition of sucrose, glucose, fructose and maltose. The crude enzyme was partially purified by a combination of ultrafiltration and ammonium sulfate precipitation. The partially purified enzyme was separated by molecular exclusion chromatography into three components A, B and C, with molar masses ranging from 13.2 to 64 kDa. Only fraction B exhibited enzymic activity and further fractionated by ion-exchange chromatography into four components I–IV. Among these components, only fractions I and II possessed transeliminase activity. Both fractions had an optimum activity at pH 8.5 and 35°C, and were stimulated by Ca²⁺, Mg²⁺, Na⁺ and K⁺ but inhibited by EDTA and DNP. The apparentK _m for the degradation of pectin by fractions I and II were 6.2 and 8.0 g/L, respectively.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

Resolution and Properties of Two High Affinity Cyclic Adenosine 3':5'-monophosphate-Binding Proteins from Wheat Germ

A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 × 10⁵ (at low ionic strength) and 2.8 × 10⁵ (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMP-binding protein (cABP I). The K_d values for cAMP are about 10⁻⁶ molar and 10⁻⁷ molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatives having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP.     

Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel–nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS–PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH₂-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues −180 to −175, NH₂-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH₂-terminal sequence, LDXNQLY, corresponding to residues −73 to −67 of the proregion peptide and thus were generated by removal of 126 residues from the NH₂-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37°C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k_cat/K_m) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM⁻1 s⁻¹, respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing 2 × 10⁹ High Five cells.