Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Problems encountered in measuring erythrocyte pyrimidine 5'-nucleotidase activity

An improved method of two-dimensional electrophoresis that allows unequilibrated first-dimension gels to be loaded and electrophoresed on second-dimension gels twice the length used in the O'Farrell technique has been developed. Normally, the electrophoresis of unequilibrated first-dimension gels on long second-dimension gels with the resolving gel set at pH 8.8 results in poor resolution of low-molecular-weight proteins. Adjusting the pH of the resolving gel to pH 8.3 maintains the low-molecular-weight proteins in a stacked configuration during their migration through the length of the 10% acrylamide gel. Utilization of a 10 to 20% exponential polyacrylamide gradient in a resolving gel set at pH 8.3 separates these low-molecular-weight proteins with excellent resolution. Electrophoretic resolution of protein spots in resolving gels set at pH 8.8 is not as sharp as in gels set at pH 8.3, and resolution progressively deteriorates in gels set at higher pH values.     

Characterization of fish acid proteases by substrate–gel electrophoresis

Several analytical techniques based upon the use of substrate–polyacrylamide gel electrophoresis were evaluated to achieve characterization of aspartate proteases in fish stomach. Since aspartate proteases of fish are more stable at high pH than mammalian pepsins, the most accurate technique for activity assessment is electrophoresis at neutral pH and revealing of such activity at low pH with hemoglobin as substrate. The technique is suitable for characterization of proteases and in comparative assessment of acid protease activity in different sparids.     

Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine-sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP-CGM). The OP-CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8 kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS-polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.     

Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

Rabbit-liver phosphorylase: improvement of the purification procedure and assay of the inactive b form

Continuous electrophoresis buffers are described for polyacrylamide gels at pH values ranging from 3.8 to 10.2. The buffers consist of an acidic and a basic component with pK values near the pH of the buffer. The pH is maintained to within 0.5 pH unit in the electrode compartments during prolonged electrophoresis. Some proteins produce clear bands on gels with each of the 10 buffers. The buffers provide an expansion of the pH range of gel electrophoresis and are likely to be useful in the detection of genetic variation in proteins and in other applications.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

两种酶谱方法在κ-卡拉胶酶活性鉴定中的应用

对分离纯化后的κ-卡拉胶酶进行SDS-PAGE电泳和酶谱试验鉴定,比较κ-卡拉胶酶活性鉴定中的两种酶谱试验方法。一种是先进行非变性PAGE电泳,电泳完毕,将电泳胶与事先准备好的底物胶叠合在一起,35℃孵育液孵育。另一种是在此试验方法基础上进行改进,直接在电泳分离胶中加入0.2%底物,进行SDS-PAGE电泳,电泳结束,用TritonX-100将电泳胶复性,孵育液孵育。试验结果表明改进后的酶谱方法操作简单,具有良好的灵敏度和精确的定位。同时,利用改进后的酶谱方法对κ-卡拉胶酶活性进行了反应时间的研究,结果显示,反应时间为8 h时,降解条带最清晰,最有利于相似分子量酶的辨别。     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.

Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50 degrees C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and beta-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85 degrees C for 20 min. Divalent metal ions Mg, Co, and Mn were required for maximum activity of the enzyme. The K(m) values for D-xylose and D-glucose at 80 degrees C and pH 7.5 were 6.66 and 142 mM, respectively, while K(cat) values were 2.3 x 10 s and 0.5 x 10 s, respectively.     

A reversal in relative mobility of the two large subunits of brine shrimp (Na+ + K+)-adenosinetriphosphatase

The two large subunits of brine shrimp Na,K-ATPase can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at neutral pH and at acidic pH. These subunits appear to reverse their positions on the gel relative to each other when resolved at acidic pH relative to neutral pH. The migration of both subunits is apparently affected by charge, even in the presence of 2.5% sodium dodecyl sulfate.     

Biochemical isolation and physiological identification of the egg- laying hormone in Aplysia californica

It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone.