50 ku keratin-like protein and β-microtublin coexist in higher plant cells

IF-like proteins have been obtained from suspension cells of Nicotiana tabacum by selective extraction. Western blot analysis shows that the major components of IF-like proteins are 6 keratin-like proteins of 64, 58, 55, 54, 50 and 45 ku. Specially the 50 ku ptotein also reacts with polyantibody against microtublin. Two-dimensional gel electrophoresis shows that the 50 ku protein is composed of two different proteins and their amino acid sequences have been determined. Part of the sequence of one protein is identical to that of β-microtublin and the other protein's sequence has no significant homologue, which should be a new sequence-unknown protein. These results suggest that 50 ku keratin-like protein and β-microtublin coexist in higher plant cells, and that may lead to the phenomenon of co-distribution of IF and microtuble in plant cells.     

Primary Structural Characterization of Phospholipid Hydroperoxide Glutathione Peroxidase

IntroductionBoth physico-chemical properties and functions ofproteins are based on their primary structure.Theoretical analysis of the primary structure ofproteins will give the structural characteristics ofthe same kind of proteins and provide ideas forfurther experimental research. More than 2 0native or deduced phospholipid hydroperoxideglutathione peroxidase (PHGPX) proteins werefound;three of which were cloned from rice,momordica and radishes in our laboratory[1 ,2 ] .There is a need to…     

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteinerich domains and localization of these proteins

The 5′-terminal (RTn) and 3′-terminal (RTc) halves of the coat protein readthrough domain and the 19 ku cysteine-rich protein of Chinese wheat mosaic virus (CWMV) were amplified by RT-PCR, cloned and expressed in E. coli. Antisera and monoclonal antibodies against these proteins were prepared by immunising these purified proteins to mice. Detection of RTn, RTc and 19 ku proteins in CWMV infected wheat sap and leaf tissue indicated that the RTn and RTc proteins were distributed on the surface of virus particles whereas the 19 ku protein was in the cytoplasm of the infected wheat cells.     

Identification and role of plasma membrane aquaporin in maize root

Using antiserum against expressed aquaporin fusion protein, GST-RD28, the distribution of aquaporin in the plasma membrane of maize root protoplasts has been examined under confocal laser scanning microscopy by indirect fluorescence staining. Results indicate that there are abundant aquaporins in maize roots, which are distributed in plasma membrane unevenly. Western blotting analysis of total protein solubilized from maize root plasma membrane shows that antiserum against GST-RD28 can cross-react with one protein around 55 Ku. Another 28 ku protein can also be detected when the concentration of SDS and DTT in SDS-PAGE sample buffer is increased. The 55 and 28 ku proteins may be dimeric and monomeric of aquaporin respectively. Functional experiments show that aquaporin blocker HgCl₂ and aquaporin antiserum can suppress the swelling of maize root protoplasts in hypotonic solution, indicating that aquaporin in plasma membrane of protoplast facilitates rapid transmembrane water flow.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

Isolation and characterization of the gene for a yeast mitochondrial import receptor

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.     

抗三尖杉酯碱的HL60细胞蛋白质磷酸化

研究了抗三尖杉酯碱的ＨＬ６０细胞蛋白质磷酸化的变化。经差速离心得到纯膜蛋白，抗性细胞有一高度磷酸化的１１０ｋｕ的蛋白质存在，免疫沉淀ｃ－ＫＡＦ－１蛋白激酶，抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，其活性被蛋白激酶Ｃ抑制剂ＣａｌｐｈｏｓｔｉｎＣ明显地抑制。结果表明：ＨＬ６０细胞对三尖杉酯碱的抗药性与蛋白质高度磷酸化有关；抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，可能与多药抗药性和抗细胞调亡有关。     

Interactions between the nuclear matrix proteins and the 5′-flankingcis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMP_k) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE II, - 446 - 419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay. Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMP_k in K562, cells may be composed of two polypeptides (∼ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA (- 392 - 177 bp) in the 5′-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.     

螅状独缩虫(Carchesium polypinum)类中间纤维的初步研究

利用间接免疫荧光和免疫印迹发现螅状独缩虫中存在两种分子质量为58ku和66ku的蛋白。这两种蛋白能分别与抗波形蛋白和抗核纤层蛋白B的抗体反应。分子质量66ku的蛋白位于大核边缘。另外,细胞经分级抽提后大核周围存在核纤层样结构。基于上述结果,可以认为螅状独缩虫中存在类中间纤维。     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.