Characterization of an Inhibitor for Lactobacillus bulgaricus in Tomato Juice

Tomato juice (serum) added to milk in high concentration caused inhibition of acid production by Lactobacillus bulgaricus. The inhibitor was partially purified by adsorption on charcoal. Further isolation and purification involved paper chromatography in two different solvent systems. Ultraviolet-absorption spectra and thin-layer chromatography were used in characterization studies. The inhibitor was found to be a xylose- and adenine-containing nucleotide.     

Characterization of an Inhibitor of Hepatic Cholesterogenesis Present in Hepatic Microsomes

An inhibitor of hepatic cholesterol synthesis present in hepatic microsomes can be solubilized either by an acetone or an ethanol powder preparation. Other methods such as methanol and chloroform:methanol powder preparations and treatment with EDTA do not solubilize the factor.

The factor appears to be proteinaceous since its activity is lost after exposure to proteolytic enzymes and heat treatment. In addition, the inhibitor does not require a phospholipid for activity.

This inhibitor is stable for long periods (60 hrs.) at room temperature and can be isolated in good yield from liver maintained at 4°C for 8 hours postmortem.     

Characterization of an aminopeptidase from Pseudozyma hubeiensis 31-B and potential applications

Yeast strain 31-B was isolated from the digestive juices of Nepenthes alata as an aminopeptidase producer and identified as Pseudozyma hubeiensis via morphological testing and comparative 26S ribosomal DNA-D1/D2 gene sequence analysis. Strain 31-B produced aminopeptidase as extracellular peptidase, but proteinase activity was not detected in the culture filtrate. The aminopeptidase from strain 31-B was purified from filtered culture medium by (NH₄)₂SO₄ precipitation and four column chromatography steps: Diethylaminoethyl (DEAE)-Toyopearl 650 M, Butyl-Toyopearl 650 M, hydroxylapatite, and Toyopearl HW-55. Sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded the purified enzyme as a single band with molecular mass 75.3 kDa. The optimum temperature and pH were approximately 40 °C and 8.0, respectively. The purified aminopeptidase preferentially hydrolyzed Leu-p-NA and its activity was inhibited by ethylenediaminetetraacetic acid. The isolated aminopeptidase reduced the bitterness of peptides generated from milk casein using a bacterial proteinase. These results show that the aminopeptidase produced by P. hubeiensis 31-B has potential application as a food additive in the dairy industry.     

凝血酶抑制剂TTI的表达及性质研究

将来源于采采蝇的TTI基因序列改造成大肠杆菌偏爱密码子,利用重组PCR方法获得TTI目的基因片段,在大肠杆菌中得到高效表达。经纯化获得了纯度高于98%的融合蛋白,建立了酶活测定方法。实验证明融合蛋白具有抑制凝血酶的活性。当凝血酶浓度为10U/ml,纯化的融合TTI体积为10 l,底物浓度为250 mol/L,融合蛋白对凝血酶的抑制率为73%,确定反应类型为竞争性抑制,Ki为35 mol/L。     

X-ray Structure Analysis and Characterization of AFUEI,an Elastase Inhibitor from Aspergillus fumigatus

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.     

Characterization and Role of an Endogenous Inhibitor in the Induction of Cold Hardiness in Acer negundo

An inhibitor extracted from short day treated Acer negundo leaves was compared to abscisic acid in 4 different solvent systems. The chromatographic properties of abscisic acid and the inhibitor were in very close agreement. Treatment of Acer negundo plants under non-hardening preconditions (long days) with either the inhibitor or abscisic acid increased hardiness after a hardening period of 3 weeks at 40°. A gibberellin-inhibitor relationship was further studied by making comparison of extracts of plants subjected to either 4 weeks of long days, long days + 5° nights, or short days. These tests indicated that gibberellin-like activity was greatest when the treatment included long days. Abscisic acid-like levels were highest when the treatments consisted of short days or long days + 5° nights. Since the latter groups are the most capable of developing hardiness, the hardening process appears to be more closely related to a build-up of abscisic acid levels than a reduction of gibberellin levels.     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

Purification and Characterization of a Rice Cysteine Proteinase Inhibitor

A proteinaceous substance that inhibited the activity of papain (EC 3.4.22.2) was found in seeds of rice, Oryza sativa L. japonica. This cysteine proteinase inhibitor (CPI) was purified by a series of purification procedures including CM-Sephadex C-50, Sephadex G-75, and DEAE- Sephadex A-50 chromatography. The CPI was a single polypeptide with a molecular weight of about 12,000, with an isoelectric point at pH 5.3. The CPI was stable below 100°C and between pH 2.2 ~ 9.0. The inhibition of papain by the CPI was non-competitive, with a Ki value of 2.44 × 10^-8 m. The complete inhibition of papain was reached by an equimolar concentration of the CPI.     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.     

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ``SSI-like proteins' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338–4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins. Received: 23 August 1996 / Accepted: 20 November 1996     

Purification and Conformational Characterization of a Novel Interleukin-2 Mutein

The Protein Journal - Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development...     

一种白细胞介素-2缓释微球制备新方法及体外表征

目的：开发一种白细胞介素-2（m-2）长效缓释微球剂型。方法：采用S／O／W法制备了白介素-2因子多糖微粒的PLGA微球,考察了微球的表面形态、粒径分布等,并且运用ELISA方法考察了微球的体外释放效果。结果：本方法制备的白介素-2因子微球光滑圆整,粒径分布较均匀,体外缓释达32天,累积释放率近90％。结论：本方法制备的白介素-2因子微球,不仅具有有效地保护IL-2蛋白活性,同时实现长效缓释的目标,是一种可行的蛋白缓释方案。     

抗人组织金属蛋白酶抑制剂—1核酶的体外制备和活性鉴定

研究了特异切割人瘢痕组织中组织金属蛋白酶抑制剂 1(tissueinhibitorofmetalloproteinases 1,TIMP 1)的锤头状核酶 ,测定了其体外切割活性。制备特异切割TIMP 1的U6snRNA嵌合型核酶基因克隆。TIMP 1mRNA基因片段克隆至T载体。用体外转录法大量制备以 [α 3 2 P]UTP标记的核酶及靶RNA ,进行体外切割实验。结果表明 :核酶∶底物 =1∶1时 ,活性的U6snRNA嵌合型核酶 (U6Rz35 8)在 5 0℃具有最佳切割活性 ,切割效率为 76 .34% ;37℃时 ,切割效率为 5 5 .2 1%。5 0℃时 ,Km=39.6nmol/L ,kcat=0 .2 1min-1;而点突变型核酶U6Rz35 8m 没有切割活性。制备的U6 Rz35 8有良好的特异切割活性 ,有望在瘢痕成纤维细胞内抑制人TIMP 1的表达。     

Cloning and Characterization of Trypsin Inhibitor Gene from Vigna unguiculata

A trypsin inhibitor, member of Bowman-Birk family, was isolated and purified from cowpea (Vigna unguiculata]). Polyclonal antibodies were raised against cowpea trypsin inhibitor (CpTI) protein in rabbits. The gene for CpTI was amplified by polymerase chain reaction and cloned in a bacterial expression vector pVCATFR18416. The expression of CpTI protein in BL21 (DE3) strain of Escherichia coli was confirmed by western blot studies. The CpTI gene was also sequenced and found to exhibit 100% homology with already published sequence of CpTI gene.     

波叶青牛胆胰蛋白酶抑制剂的纯化及其性质研究

采用亲和层析及电泳制备等方法 ,从防已科植物波叶青牛胆 (Tinosporacrispa)根茎中分离到一种胰蛋白酶抑制剂TCTI。对其性质研究表明 :TCTI的相对分子量约为 10 0kD ,对牛胰蛋白酶的抑制常数Ki为 2 0 8× 10 7mol/L (BAPNA为底物时 ) ,摩尔抑制比为 1∶3 5 ,是一种抑制活力很强的竞争性抑制剂。TCTI具有很高的耐热性 ,在 10 0℃加热 60min ,仍保持 93%的抑制活力。另外 ,TCTI在低温容易结晶     

Molecular Dissection of Human Interleukin-31-mediated Signal Transduction through Site-directed Mutagenesis

Interleukin (IL)-31 is a recently described cytokine, preferentially produced by T helper 2 lymphocytes and associated with skin diseases, such as atopic dermatitis. IL-31 is a member of the four α-helix bundle cytokine family and is related to the IL-6 subgroup. Its heterodimeric membrane receptor is composed of the gp130-like receptor (GPL) subunit associated to the oncostatin M receptor subunit. We identified critical amino acids implicated in the ligand receptor interaction by computational analysis combined with site-directed mutagenesis. Six IL-31 residues selected for their putative involvement in cytokine receptor contact sites were alanine-substituted, and the corresponding proteins were expressed in mammalian and bacterial systems. Biochemical, membrane binding, cell signaling, and cell proliferation analyses showed that mutation E44A, E106A, or H110A abolished IL-31 binding to GPL and the subsequent signaling events. A second ligand receptor-binding site involved Lys¹³⁴, with alanine substitution leading to a protein that still binds GPL, but is unable to recruit the second receptor subunit and the subsequent signaling pathways. The results indicate that IL-31 recognizes its receptor complex through two different binding sites, and we propose a three-dimensional model for IL-31.