Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Autolysis of phospholipids in homogenates of various plant tissues

The rates of autolysis of endogenous phospholipids were measured in homogenates of 17 cultivated plant tissues. For seven out of the 17 samples, the rates of autolysis of phosphatidylcholine (PC) were greater than 10% hydrolysis per hr (at pH 7.5 and 4°). The addition of dibucaine (2 mM) inhibited the autolytic rates in homogenates of 12 out of 17 samples. The only plant homogenates where dibucaine stimulated the autolytic rates were those of potato tubers. Although dibucaine inhibited the rate of autolysis of PC in potato leaf homogenates there was no advantage to adding it during homogenization and preparation of differential centrifugation fractions from potato leaves. Homogenization with different types and concentrations of osmotica affected the rates of autolysis of PC. Buffering the homogenates at pH 8 drastically inhibited the rates of autolysis in potato leaf homogenates but had little effect on bean leaf homogenates. Various strategies for controlling the rate of membrane lipid breakdown in different plant extracts are discussed.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.     

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie brilliant blue G-250

A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

A simple,rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation

The Multiskan spectrophotometric system and Coomassie brilliant blue G-250 protein-dye binding method have been used together to measure NaOH-solubilized protein in subcellular membrane fractions prepared from isolated rat adipose cells. Forty-eight samples can be read in duplicate within 1 min. Sucrose in concentrations up to 0.7 m interfere only moderately with the assay. A rapid and convenient method is, therefore, now available for multiple protein determinations following sucellular fractionation on sucrose gradients.     

Purification and Some Properties of the Nitrogenase From Soybean (Glycine max Merr.) Nodules

The nitrogenase system in cell-free extracts of soybean nodule bacteroids was fractionated into 2 components by use of protamine sulfate or polypropylene glycol precipitation followed by chromatography on DEAE-cellulose. Iron and molybdenum were concentrated in 1 fraction and iron in the other. Combination of fractions resulted in a striking stimulation in activity relative to the activity of individual fractions. The effect of different proportions of the 2 fractions on specific activities was studied. The ratios of the rates of reduction of acetylene and N₂ by extracts or fractions of different purities were relatively constant. Extracts or fractions retained most of their nitrogenase activities when stored in liquid N₂.     

家蚕丝腺蛋白质组学研究方法的建立

通过高精度的双向电泳技术对家蚕中部丝腺组织的蛋白质进行分离,采用基质辅助激光解析电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)对其中一些表达量较高的蛋白点进行鉴定,并利用GPMAW(GeneralProtein/MassAnalysisforWindows)软件结合家蚕基因组预测的蛋白质数据库构建本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行分析。研究发现,经过双向凝胶电泳及其图象分析技术,硝酸银染色和考马斯亮蓝染色分别能分离出500个以上和100个以上的蛋白点。这些蛋白质点主要集中在分子量15~90kD区域,等电点pH3·5~7之间。MALDI-TOF-MS鉴定的25个考染蛋白点中有60%以上的PMF(PeptideMassFingerprint)的信号峰较强。在数据库检索过程中,利用家蚕肽质量指纹数据库所得检索结果与在Mascot的检索结果相比,前者不仅能够准确鉴定出一些已有研究报道的蛋白,从而验证检索方法的可行性,而且还能够对一些已经被家蚕基因组数据库所预测但未曾报道的新蛋白质进行鉴定,从而建立了一整套适合于家蚕蛋白质组研究的方法,并为其它绢丝昆虫蛋白质组研究提供了重要参考。     

Autolysis of Lactococci: Detection of Lytic Enzymes by Polyacrylamide Gel Electrophoresis and Characterization in Buffer Systems

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.     

A comparative study of the protein content of some helminths and the suitability of assay methods

The protein content of fresh homogenates and their corresponding TCA precipitated fractions of 10 different species of helminths was estimated by the methods of Lowry et al. and Spector using the Folin phenol reagent and Coomassie brilliant blue G-250 respectively. The former method gives exaggerated values as compared to the latter method. The parasite phenols, phenolic proteins and catecholamines could be responsible for interference in the Lowry's procedure. The TCA noln-precipitable moieties also give colour only with the Folin phenol assay. The pronounced intra-specific differences in the total protein content of helminths reflect their metabolic variations and adaptations. Habitat does not appear to influence the protein content of parasites, however, the effect of host variation was evident in the pouched amphistome G. crumenifer. It is concluded that the dye binding method gives more consistent results and it can be conveniently applied to crude tissue homogenates of helminths.     

The antenna system of Rhodospirillum rubrum. Detection of macromolecular constituents not stainable by Coomassie brilliant blue in solubilized preparations of the B880 complex

A solubilized preparation of the major Rhodospirillum rubrum antenna complex (B880) was obtained by a described procedure and its polypeptide composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only two polypeptides of molecular weights close to 7000 were detected after staining the gels with Coomassie brilliant blue. However, several other constituents could be visualized by silver staining or by an immunochemical method. When the preparation was chromatographed on Sephacryl, some of the resulting fractions exhibited the characteristic B880 absorption spectrum and contained only the two proteins that were detectable with Coomassie brilliant blue. In those fractions the A ₂₈₀/A ₈₈₀ratio was 0.4, which indicated a significant improvement of the bacteriochlorophyll to protein ratio over the unchromatographed preparation (A ₂₈₀/A ₈₈₀=0.7). Other chromatography fractions lacked bacteriochlorophyll and contained a carotenoid which seemed to be bound to protein. The macromolecular constituents present in these latter fractions differed from those associated to the purified B880 complex in their electrophoretic moblities and/or in their staining properties. That suggested the possible existence of a carotenoprotein that did not result from the B880 complex upon loss of bacteriochlorophyll.     

Effects of temperature on the autolytic enzyme system of Streptococcus faecalis.

The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.     

The rate of initiation of alpha- and beta-globin mRNA translation is modulated by 50 kDa, 28-kDa and 24-kDa polypeptide-containing fractions

The relative rates of initiation of alpha- and beta-globin mRNA translation in a Krebs II ascites cell-free system are differently modulated by a 50-kDa protein and two fractions containing either a 28-kDa or a 24-kDa polypeptide. Each of these fractions stimulated a discrete step that limits initiation of protein synthesis, but other rate-limiting steps take place upstream and/or downstream, resulting in characteristic kinetics of the stimulation of alpha- and beta-globin synthesis. The ascites extracts appear to be deficient in these activities.     

Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies.

Antigens of Mycobacterium tuberculosis found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.     

Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils

Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for protein concentration determination. In this study, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CB to lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the native protein and to amyloid fibrils but gives distinctly different spectral responses. The absorption and fluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different, and this was most likely due to differences in secondary structure as monitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.     

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.