PURIFICATION AND CHARACTERIZATION OF THE 20S PROTEASOME FROM THEALGA CHARA CORALLINA (CHAROPHYCEAE)1

We purified the 20S proteasome from the alga Chara corallina Willd with DEAE–ion‐exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme bynondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two‐dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin‐like, trypsin‐like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin‐like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin‐like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin‐like activity was inhibited by N‐ethylmaleimide, p‐chloromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin‐like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.     

芝田硫化叶菌ssh7a和ssh7b基因在大肠杆菌中的表达及重组蛋白的性质

极端嗜热古菌－－芝田硫化叶菌ＤＮＡ结合蛋白Ｓｓｈ７ａ和Ｓｓｈ７ｂ的编码基因（ｓｓ７α和ｓｓｈ７ь）在大肠杆菌中得到表达。量均达到细胞蛋白总量的１０％￣１５％。重组蛋白通过一个包括热处理步骤的简单纯化程序得到纯化。重组Ｓｓｈ７ａ和Ｓｓｈ７ｂ与松弛及负超螺旋ＤＮＡ的结合与天然Ｓｓｈ７蛋白无异,与天然Ｓｓｈ７相似,Ｓｓｈ７ａ在与ＤＮＡ结合时能免固定负超螺旋,每固定一个负超螺旋约需２２个Ｓｓｈ７ａ分子。这     

水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性

在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。     

ISOLATION, CHARACTERIZATION, AND CHROMOSOME MAPPING OF AN ACTIN GENE FROM THE PRIMITIVE GREEN ALGA, NANNOCHLORIS BACILLARIS (CHLOROPHYCEAE)

Historically, the genus Nannochloris has been classified using the morphology of cell division, although the mechanics of division remain relatively poorly understood. Nannochloris bacillaris reproduces by binary fission. Microscopic observation with fluorescein isothiocyanate-phalloidin showed that actin filaments localized near the nucleus and appeared as a ring- or beltlike structure in the septum-forming area in the middle of the cell during cell division. In primitive unicellular Chlorophyta such as N. bacillaris, actin is also thought to play important roles in nuclear migration and cell division. The N. bacillaris actin gene has three exons and two introns defined by two exon–intron junctions with splice site consensus sequences. The two introns are located at codons specifying amino acids 3/4 and 47/48. One of these, intron position 3/4, is conserved in the actin gene of Saccharomyces cerevisiae. The actin gene product was predicted to be 378 amino acids long with an estimated molecular weight of 42 kDa. There is only one copy of the actin gene in the N. bacillaris genome. Nannochloris bacillaris has 14 chromosomes that range in size from 230 kb to 3000 kb, and the total size of the genome was estimated to be 20.3 Mb. The actin gene is on either chromosome XI or XII. In a phylogenetic tree based on the actin gene sequence, N. bacillaris diverged before the divergence of Volvox, Chlamydomonas, and higher plants, and very shortly after the radiation of the Rhodophyta.     

PARTIAL CLONING AND CHARACTERIZATION OF THE cDNA OF GENERAL ODORANT BINDING PROTEIN 1 GENE IN THE ANTENNA OF HELICOVERPA ARMIGERA (HÜBNER)*

Abstract Using RT‐PCR and RACE techniques, part of the cDNA encoding the general odorant binding protein 1 gene (named as GOBP1‐Harra) from the antenna of Helwoverpa armigera (Hubner) has been cloned. The cDNA length of GOBP1‐Harm is 876 bp. The results of sequencing and structural analysis showed that the mature protein reading frame of GOBPl‐Harm is 435 base pairs in length and 145 amino acids encoded. The predicted MW and pl are 17.0 kD and 4.89, respectively. The deduced amino acid sequence showed a highly similarity to the sequence of GOBP1 from different moth species and shared several common structural features with odorant binding proteins from other insects.     

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A PEPTIDOGLYCAN RECOGNITION PROTEIN FROM THE COTTON BOLLWORM,Helicoverpa armigera

Peptidoglycan recognition proteins (PGRPs) specifically bind to peptidoglycans, and play crucial roles as pattern recognition receptors (PRRs) in mediating innate immune responses. In this study, we identified and characterized a PGRP (HaPGRP‐D) from the cotton bollworm, Helicoverpa armigera. Sequence analysis indicated that HaPGRP‐D is an amidase‐type PGRP. Expression of HaPGRP‐D was upregulated in the hemocytes of H. armigera larvae after injecting Gram‐negative Escherichia coli, Gram‐positive Staphylococcus aureus, or chromatography beads. To test the biological activity of HaPGRP‐D, purified recombinant protein was prepared. Subsequent analysis showed that rHaPGRP‐D (i) could bind and agglutinate Gram‐negative E. coli and Gram‐positive S. aureus in a zinc‐dependent manner, (ii) functioned as an amidase to degrade peptidoglycans in the presence of Zn²⁺, (iii) strongly inhibited the growth of E. coli and S. aureus in the presence of Zn²⁺, (iv) could bind to the surface of hemocytes, (v) increased the phagocytosis of E. coli cells by hemocytes in vitro, and (vi) promoted hemocyte encapsulation on chromatography beads in vitro. These results suggest that HaPGRP‐D plays important roles as PRR, amidase, and opsonin in H. armigera humoral and cellular immune responses.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF A CALCIUM‐DEPENDENT ALKALINE PHOSPHATASE FROM THE CYANOBACTERIUM ARTHROSPIRA PLATENSIS 1

In the present study, Triton X‐114 (TX‐114) is used to extract and partially purify alkaline phosphatase (ALP) from a membranous fraction of Arthrospira platensis Gomont containing cell wall, plasma membrane, thylakoids, and sheath. TX‐114 has a double effect: solubilizing cell components to liberate the enzyme and, after phase partitioning, removing chl and other pigments present in the crude extract. The recovery of the enzyme in the aqueous phase suggests the overall hydrophilic character of this enzyme. ALP was kinetically characterized at pH 11.0 using p‐nitrophenyl phosphate as substrate, giving a K_m value of 1.7 mM. Orthovanadate was seen to be a competitive inhibitor of ALP, with a K_i of 0.8 mM. The enzyme was almost completely inactivated in the presence of 70 μM EDTA, although the addition of Ca²⁺ reverted this inactivation; these results indicate that ALP from A. platensis is a calcium‐dependent metalloenzyme. When the effect of Ca²⁺ was investigated in detail, a value of 0.067 μM^?1 for the affinity constant was obtained. The enzyme was histochemically localized in the cytoplasm, cell wall, and sheath using the enzyme‐labeled fluorescent substrate (ELF) method. It is assumed that the same enzyme is either soluble in the cytoplasm and in some way “trapped” in the cell wall or in the sheath. ALP localization within the sheath and the subsequent release of phosphorus (P) may benefit the neighboring cells surrounding this layer.