Analytical isotachophoresis in the analysis of synthetic peptides.

Two peptides, one undecapeptide and one decapeptide, have been synthesized by the solid phase method. Isotachophoresis has been used as an analytical tool to guide the purification of the peptides. This technique gives qualitative as well as quantitative information about the purification progress. Furthermore, the purity and identity of the final product can be established. The method is rapid, reproducible and easy to perform. Since isotachophoresis also can be used for amino acid analysis, it might have a wide use in peptide chemistry.The two peptides, having the primary sequences around the cross-linking site of fibrin, have been tested in vitro as inhibitors of the fibrin cross-linking. Both peptides were essentially inactive.     

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.     

Improved techniques for scoring TLC plates.

A method is described for the rapid purification of acid deoxyribonucleases. It primarily involves chromatography of the enzyme-containing preparation through a DNA-ECTEOLA-cellulose column. This column retains the DNAse activity but not the inactive protein, and consequently, high degrees of purification are attained.     

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-1. [Tyr47, Nle53] preprorenin-(47-60) peptide methyl ester

The [Tyr47, Nle53] preprorenin (47-60) peptide methyl ester, a tetradecapeptide related to the human renin prosegment, has been synthesized using a three-segment coupling strategy. Selective deprotection of the segments before coupling allowed an easy removal of the final tetradecapeptide side chain-protecting groups by acidolysis and an easy purification. Antibodies raised against this peptide bound the plasmatic inactive renin.     

Occupancy of phosphorylation sites in pyruvate dehydrogenase phosphate complex in rat heart in vivo. Relation to proportion of inactive complex and rate of re-activation by phosphatase.

The [gamma-32P]ATP-back-titration method of estimating occupancy in vivo of the three phosphorylation sites in the pyruvate dehydrogenase complex was improved in precision by specific analysis with trypsin/formic acid, by more effective prevention of site-2 dephosphorylation during purification with NaF, and by other refinements. Disproportionation of phosphorylated complexes during purification was excluded. With this improved method it was shown that the relationship between occupancy of sites and the proportion of complex in the inactive form in rat heart in vivo is closely similar to that measured directly in heart mitochondria by incorporation of [32P]Pi. In the heart in vivo (as in mitochondria), occupancy of site 1 correlated linearly with the proportion of inactive complex. Occupancy of sites 2 and 3 only approached equivalence to that of site 1 when 99% of the complex was inactive (starved or diabetic rats). When 70% or less of the complex was inactive (resting or exercising fed normal rats), occupancy of sites 2 and 3 was minimal (3 less than 2) relative to site 1. The initial rate of re-activation by phosphatase of phosphorylated complex from hearts of resting or exercising fed normal rats was approximately three times that of complex from 48 h-starved rats.     

Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.     

Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial, and stromal cell lines in culture

A number of single-cell-cloned cell lines have been used to examine the growth-promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary-derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial-like cells. Epidermal growth factor (EGF) has a widespread but variable growth-stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 micrograms/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial-like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.     

Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ～106 Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Coenzyme B-12-dependent reactions. Part IV. Observations on the purification of ethanolamine ammonia-lyase.

Purification of ethanolamine ammonia-lyase (EC 4.3.1.7) from a Clostridium sp. grown at the University of Sussex, U.K. and the National Institutes of Health, U.S.A., has been compared and an improved isotopic assay for the enzyme has been developed. Successful purification of this enzyme from Sussex-grown cells requires modification of the published procedure (Kaplan and Stadtman (1968) J. Biol, Chem. 243, 1787-1793) principally a 70% decrease in volume during precipitation with 0.4 M NaCl. This modification also increases the yield from N.I.H.-grown cells. Purified enzyme, resolved of inactive cobalamins, has the same high specific activity from both sources and behaves in the same way on disc gel electrophoresis. Sussex enzyme, before resolution, has less than 20% of the specific activity of unresolved N.I.H. enzyme and contains over 50% more inactive cobalamin. The bound cobalamin from both preparations has been identified as a "base-on" Co11 psi-cobalamin.     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.     

Purification of the messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane.

The mRNA for the lipoprotein of the Escherichia coli outer membrane has been purified to 85% homogeneity. The purification procedure involved phenol extraction, NaCl extraction, gel filtration on Sephadex G-100 and Sephadex G-200, and reversed-phase column chromatography on RPC-5. The purity of the final product was estimated to be 85% by analysis of the ribonuclease T1 fingerprint of the mRNA. The purified mRNA was able to direct the synthesis of cross-reactive material with antilipoprotein serum in both the E. coli and the wheat germ cell-free protein-synthesizing systems. The size of the mRNA was determined to be 8.2 S from its mobility in polyacrylamide--agrose gels. During the purification, two other RNA species, similar in size to the lipoprotein mRNA, were also isolated. Their sizes were determined to be 8.7 and 9.1 S. They both were inactive in an E. coli cell-free protein-synthesizing system.     

Purification of a completely inactive renin from hog kidney and identification as renin zymogen

Hog renal inactive renin was separated from active renin and completely purified to an electrophoretically homogeneous state by using a new procedure which consisted of affinity chromatography on pepstatin-Sepharose, octyl-Sepharose, Affil-Gel blue and Con A-Sepharose columns, ion exchange chromatography and gel filtration. By this method a 3,000,000-fold purification was obtained with a 6% recovery from a crude kidney extract. This pure preparation was totally inactive and underwent marked activation by trypsin. It is a glycoprotein as judged by affinity to concanavalin A and has an apparent molecular weight of 50,000 as determined by gel filtration on Sephadex G-100. Treatment of the inactive renin with guanidine, urea and Triton X-100 did not cause activation indicating that the inactive renin isolated in the present study is not a product of renin-inhibitor complex.     

Purification of a form of mouse liver UDP glucuronosyltransferase which glucuronidates androgens

A form of UDP glucuronosyltransferase active in the glucuronidation of the androgens, testosterone, androsterone and dihydrotestosterone has been purified to apparent homogeneity as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis from the livers of phenobarbital-treated C57BL/6N mice. This UDP glucuronosyltransferase is inactive towards estrone as substrate. Data from chromatofocusing and purification experiments suggest that testosterone and androsterone are glucuronidated primarily by this enzyme form and to a lesser extent by an enzyme form which has a slightly higher isoelectric point. However, this major form is only responsible for about half the capacity to glucuronidate dihydrotestosterone.     

区带超速离心提纯流感病毒的初步探讨

在制备灭活流感疫苗中,采用两次蔗糖速率区带超离心从流感病毒感染的鸡胚尿液中提纯流感病毒是较简便、实用的方法,根据流感病毒分子特性设计了超离梯度并采用适当试验条件,获得了大量提纯流感病毒的初步结果。经血凝、电镜、蔗糖浓度、浮力密度等检验证明此方法是可行的。     

Purification of sarcoplasmic reticulum vesicles through their loading with calcium phosphate

A technique of purification of sarcoplasmic reticulum was devised through selective loading of the vesicular material with calcium phosphate. In presence of amount of disposable calcium lower than the maximum accumulation capacity of the total vesicular population, we have defined conditions of loading which allow the selection by centrifugation. The results described in this work show that about 30% of the starting material can be isolated as a vesicular population homogenous on the stand of the amount of accumulated cation. The purification is achieved by the removal of calcium by dissociation of the precipitate. Freeze-fracture electron microscopy studies show that the more active fraction when freed of calcium phosphate precipitate displays smooth convex (EF_s) and particulated concave (PF_p) fracture planes. It has been verified that the purification described in this work allows the removal of all the inactive material. The rate of calcium uptake of the selected preparation is about twice as large as that displayed by the starting material. The structural homogeneity of this material and the increase in the activity are good evidence for the purity of the selected sarcoplasmic reticulum vesicles.     

Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells

Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap.     

Immunological characterization of bovine lysyl oxidase

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.