Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate

A method is described for the assay of proteolytic activity, based on the digestion of L-[4,5-3H]leucine globin. L-[4,5-3H]Leucine was incorporated into the substrate at the stage of haemoglobin biosynthesis, using rabbit erythrocytes. Assay methods for proteolytic enzymes have been based on the digestion of haemoglobin, serum albumin or casein, and the determination of the trichloroacetic acid-soluble products [1,2]. More sensitive methods have been developed by using haemoglobin labelled with a fluorescent [3-5] or radioactive marker [6,7]. These methods avoid the errors which beset the Anson procedure, such as interference by impurities (purines at 280 nm and reducing compounds at 700 nm) [8]. However, methods using labelled proteins as a substrate present a number of problems, the most troublesome of which are the high blank values and the use of non-physiological substrates when chemically modified proteins are employed. In the present communication a simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5-3H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products.     

Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H<Subscript>2</Subscript>O<Subscript>2</Subscript> exposure

Summary. The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.     

Pharmacological studies on the sedative and hypnotic effect of salidroside from the Chinese medicinal plant Rhodiola sachalinensis

This is the preliminary study of sedative and hypnotic activity of salidroside (a major component of Rhodiola sachalinensis) in mice by using synergism with pentobarbital as an index for the hypnotic effect. Loss of the righting reflex was used to determine the start of sleep. Sleep latency and sleeping time were evaluated in this experiment. The results showed that salidroside could obviously shorten the sleep latency and prolong the sleeping time of mice produced by pentobarbital sodium (55 mg/kg, i.p.). Salidroside produces significant sedative-hypnotic effect. The dose-effect relationship is remarkable.     

<Emphasis Type="Italic">N</Emphasis>-Acyl derivatives of Asn,new bacterial <Emphasis Type="Italic">N</Emphasis>-acyl <Emphasis Type="Italic">D</Emphasis>-amino acids with surfactant activity

Summary. New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.     

Mobilization of Reserve Proteins During Early Stages of Seed Germination in Fagopyrum Esculentum

Hydrolysis of 13S globulin, the main storage protein in grains of common buckwheat (Fagopyrum esculentum Moench), proceeds in at least two phases during germination. The first stage, involving a limited proteolytic cleavage of the protein, is associated with increased activity of proteases having maximum activity at pH 7.6. The second stage, involving further hydrolysis of the partially cleaved protein, starts after 12 h of imbibition. During this phase, activity of proteases increased and activity maximum shifted to pH 5.6. Nevertheless, 13S globulin retains its antigenic identity till the emergence of radicle and plumule. Thus, it may not be the major source of amino acids utilized by the germinating seed during the initial stages of imbibition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Autodegradation of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

一株羽毛降解菌产酶特性的初步研究

通过富集培养从土壤中分离到一株能降解羽毛角蛋白的芽孢八叠球菌（编号为GIMN1.015）。以天然羽毛为底物,初步研究了温度、起始pH、辅助碳源以及羽毛底物含量对该菌株的蛋白酶水解活性的影响。结果表明,在羽毛发酵培养基中,菌株GIMN1.015在初始pH 11.0、温度30℃时,蛋白酶活力最强;与培养基中只含有羽毛的发酵过程相比,添加葡萄糖有利于提高蛋白酶的活性;底物浓度为1.5%时蛋白酶活性最高。本试验结果为进一步利用角蛋白降解微生物实现羽毛角蛋白的资源化利用奠定了基础。     

Synthesis and antitumor activities of 3-modified 2-methoxyestradiol analogs

The syntheses of 2-methoxyestradiol analogs with modifications at the 3-position are described. The analogs were assessed for their antiproliferative, antiangiogenic, and estrogenic activities. Several lead substituents were identified with similar or improved antitumor activities and reduced metabolic liability compared to 2-methoxyestradiol.     

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.     

The major chromoblastomycosis fungal pathogen, Fonsecaea pedrosoi, extracellularly releases proteolytic enzymes whose expression is modulated by culture medium composition: implications on the fungal development and cleavage of key's host structures

We investigated the possible secretion of peptidases by F. pedrosoi, when conidial cells were cultured in two distinct media. Aspartyl proteolytic activity was detected on the Czapeck-Dox-derived supernatant, which was blocked by pepstatin, and only active in extremely acidic conditions. The supernatant obtained after conidia growth in Kauffman medium presented metallopeptidase activity, which was active over a broad pH range and sensitive to 1,10-phenanthroline and EGTA. Additionally, both culture supernatants were able to cleave a wide range of proteinaceous substrates, including important human serum proteins (e.g. albumin and immunoglobulin G) and extracellular matrix components (e.g. fibronectin and laminin). As peptidases participate in different cellular metabolic pathways, we also tested the influence of proteolytic inhibitors on the F. pedrosoi conidia development in vitro. The metallopeptidase inhibitors, 1,10-phenanthroline, EGTA and EDTA, strongly abrogated the growth of conidial forms by approximately 95%, 85% and 60%, respectively. Moreover, 1,10-phenanthroline blocked the differentiation process from conidia to mycelia, an essential step during the F. pedrosoi life cycle. Phenylmethanesulfonyl fluoride, a serine peptidase inhibitor, slightly reduced the conidial growth, whereas proteolytic inhibitors of cysteine (E-64) and aspartic (pepstatin) type peptidases did not alter conidial developmental behavior. In summary, our results showed for the first time the expression of extracellular proteolytic activity by F. pedrosoi conidial cells.     

A simple procedure for the detection of plant extracellular proteolytic enzymes

A simple procedure for the detection of extracellular plant proteolytic enzymes using insoluble dye stained gelatin substrates incorporated into an appropriate culture medium is described. Extracellular proteinases produced by the tested plant cells (callus culture and cell suspension) hydrolyzed the substrates and dyed peptide fragments were released. Dyed zones around and under the proteinase-producing callus cultures were formed on the agar medium. Similarly, coloration of the culture media using proteinase-producing cell suspensions was observed. This revised version was published online in August 2006 with corrections to the Cover Date.