High performance liquid chromatography,chemical laboratory practice: By Heinz Engelhardt. Springer-Verlag,New York/Berlin, 1979. 248 pp. $29.80

A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, $\text{v}\text{v}$). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more.     

Changes in the capsid structure and stability of defective particles of bacteriophage R17.

Serological and chemical methods were used to compare the capsid structure and stability of R17 phage and amA31 defective particles. Immunodiffusion analysis demonstrated identity between intact R17 and amA31 capside and between dissociated subunits of both R17 and amA31 and purified coat protein. Radioimmunoassays detected an antibody in R17 antisera that binds to intact R17 but could not be absorbed from R17 antisera with amA31. The R17 antibody remaining in amA31-absorbed sera did not neutralize infectivity of R17 phage. Differences between the surface composition of R17 and amA31 capsids were also detected by iodination. Capsids of R17 bound approximately four times more 125I than amA31, which was accounted for by a decreased 125I labeling of coat protein. Finally, amA31 capsids dissociated under milder conditions of sodium dodecyl sulfate treatment than R17 capsids. The sodium dodecyl sulfate dissociation of both R17 and amA31 capsids resulted in the formation of a transient 38,000-dalton intermediate, which subsequently dissociated to coat protein monomers. Preparations of dissociated R17 capsids also contained assembly protein was also found in preparations of dissociated amA31 capsids.     

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1.

We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis.

Isolated membrane fractions of Escherichia coli K-12 yielded complex immunoprecipitate patterns when Triton X-100 and sodium dodecyl sulfate extracts were examined by crossed immunoelectrophoresis with antienvelope immunoglobulins. Twelve of the 46 antigens in the immunoprecipitate patterns of inner (plasma) membranes were identified by zymograms and/or by the use of specific antisera. The following enzyme activities were detected in immunoprecipitates: 6-phosphogluconate dehydrogenase (EC 1.1.1.43); adenosine triphosphatase (EC 3.6.1.3); glutamate dehydrogenase (EC 1.4.1.4), two separate components; malate dehydrogenase (EC 1.1.1.37); dihydroorotate dehydrogenase (EC 1.3.3.1); succinate dehydrogenase (EC 1.3.99.1); lactate dehydrogeanse (EC 1.1.1.27); reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3); protease (EC 3.4.21.1); and glycerol 3-phosphate dehydrogenase (EC 1.1.99.5). The corresponding immunoprecipitate pattern for isolated outer membranes consisted of at least 25 discrete antigens and differed strikingly from that obtained with inner membranes. Two major immunogens were identified as lipopolysaccharide and Braun lipoprotein. A protease-active immunoprecipitate was also detected in this fraction, but attempts to identify the Rosenbusch matrix protein in the crossed immunoelectrophoretic profile were unsuccessful.     

A two-cycle immunoprecipitation procedure for reducing nonspecific protein contamination.

A two-cycle immunoprecipitation procedure is described that markedly reduces nonspecific protein contamination occurring during the precipitation of hepatic lipase from rat H4 hepatoma cells. In this method, the precipitation of immune complexes during both cycles is achieved by utilizing a sodium dodecyl sulfate (SDS)-washed preparation of lyophilized Staphylococcus aureus cells (Staph A); this washed preparation effectively removes Staph A contaminants without compromising the ability to bind immune complexes. Following initial immunoprecipitation of the antigen, the Staph A/IgG/antigen complex containing coprecipitated nonspecific proteins was dissociated with SDS. Triton X-100 was added to the dissociated immunoprecipitate at a concentration (by weight) of at least 5 parts Triton X-100 to 1 part SDS. A second cycle of immunoprecipitation was then initiated by addition of fresh antibody, followed by Staph A precipitation of immune complexes and analysis by SDS-polyacrylamide gel electrophoresis. The two-cycle procedure is shown to be reproducible and suitable for the quantitative determination of relative amounts of hepatic lipase. The procedure described here is generally applicable to the immunoprecipitation of other antigens.     

Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.     

Large Tandem,Higher Order Repeats and Regularly Dispersed Repeat Units Contribute Substantially to Divergence Between Human and Chimpanzee Y Chromosomes

Comparison of human and chimpanzee genomes has received much attention, because of paramount role for understanding evolutionary step distinguishing us from our closest living relative. In order to contribute to insight into Y chromosome evolutionary history, we study and compare tandems, higher order repeats (HORs), and regularly dispersed repeats in human and chimpanzee Y chromosome contigs, using robust Global Repeat Map algorithm. We find a new type of long-range acceleration, human-accelerated HOR regions. In peripheral domains of 35mer human alphoid HORs, we find riddled features with ten additional repeat monomers. In chimpanzee, we identify 30mer alphoid HOR. We construct alphoid HOR schemes showing significant human–chimpanzee difference, revealing rapid evolution after human–chimpanzee separation. We identify and analyze over 20 large repeat units, most of them reported here for the first time as: chimpanzee and human ~1.6 kb 3mer secondary repeat unit (SRU) and ~23.5 kb tertiary repeat unit (~0.55 kb primary repeat unit, PRU); human 10848, 15775, 20309, 60910, and 72140 bp PRUs; human 3mer SRU (~2.4 kb PRU); 715mer and 1123mer SRUs (5mer PRU); chimpanzee 5096, 10762, 10853, 60523 bp PRUs; and chimpanzee 64624 bp SRU (10853 bp PRU). We show that substantial human–chimpanzee differences are concentrated in large repeat structures, at the level of as much as ~70% divergence, sizably exceeding previous numerical estimates for some selected noncoding sequences. Smeared over the whole sequenced assembly (25 Mb) this gives ~14% human–chimpanzee divergence. This is significantly higher estimate of divergence between human and chimpanzee than previous estimates.     

Isolation and characterization of a new fimbrial antigen (CS1541) from a porcine enteroxigenic Escherichia coli O8: KX105 strain

Abstract A fimbrial antigen (CS1541) was extracted and purified from the porcine enterotoxigenic Escherichia coli strain 1541P (O8:K-:H9). CS1541 fimbriae appeared as long thin filaments 3–5 mm in diameter. CS1541 antigen consisted of two peptide bands of about 18 and 19 kDa as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. It was expressed both at 37°C and 15°C and did not demonstrate hemagglutinating properties. It was antigenically distinct from the fimbrial antigens K88, K99, F41, FY(Att25), F165, type 1, CFA-I, and CFA-II complex but demonstrated serological cross-reactions with the 987P fimbrial antigen.     

Immunochemistry of phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (σ₂₀₀) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a σ₂₀₀ value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

The glucocorticoid receptor (GC-R) isolated from the mouse AtT-20 pituitary tumor cell line exists in three forms. The untransformed (non-DNA-binding), 9.1S species (319K) can be converted into two transformed (DNA-binding) species. One of these (5.2 S, Mr 132K) appears to be composed of one molecule of the hormone-binding, monomeric protein (96K) plus a small RNA, while the second transformed species is the monomeric, hormone-binding subunit (3.8 S, 96K) itself. We wished to determine whether the untransformed GC-R contains RNA or if the monomer binds to RNA subsequent to subunit dissociation (which occurs during receptor transformation). Kinetic studies using both the crude and purified untransformed GC-R show that the untransformed, 9.1S GC-R dissociates into 3.8S monomeric subunits, without forming a transient 5.2S complex. The untransformed receptor was then purified with affinity chromatography, gel filtration, and DEAE-cellulose chromatography. One major protein band, corresponding in size to the GC-R monomer (94K-96K), was observed on sodium dodecyl sulfate-polyacrylamide gels upon silver staining or fluorography of [3H]dexamethasone mesylate covalently labeled receptor. In vivo 32P-labeling of AtT-20 cells, followed by purification of the untransformed GC-R, yielded two major 32P-labeled components (94K-96K and 24K). Both of these bands were protease-sensitive, contained phosphoserine, and were unaffected by ribonuclease treatment. We conclude that the untransformed mouse GC-R is wholly proteinaceous and contains no RNA. Thus, RNA binding occurs subsequent to dissociation of the oligomeric, untransformed GC-R complex into monomers.     

Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids : characterization and reconstitution into liposomes

We have used the nonionic detergent octyl-β-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P₇₀₀, and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI^*, is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

Identification, Purification, and Characterization of Two Forms of Serotonin Binding Protein from Rat Brain

Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K from of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by--SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants--56K form showed higher affinity for 5-HT (KD1 = 0.4 nM; KD2 = 32 nM), whereas the 45K form showed lower affinity (KD1 = 9.7 nM; KD2 = 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity--56K (19:1), 45K (10:1); (3) isoelectric point--the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration.

The binding of salicylate ion to human serum albumin (HSA) was studied in 100 mM potassium phosphate buffer (pH 7.4, 25 degrees C), using equilibrium dialysis and fluorescence titration methods. The protein samples tested were (a) dialyzed human plasma and (b) a commercial preparation of HSA, essentially free of globulin and fatty acids. Independent of the analytical method used, Scatchard and nonlinear regression analyses of the data pointed to a single class of high-affinity salicylate binding sites. On the other hand, the binding parameters were found to be method dependent. K(d) ranged between 25 +/- 2.4 and 62 +/- 15 microM in equilibrium dialysis and between 10 +/- 1.3 and 40 +/- 3.0 microM in fluorescence titration. (The higher limits refer to plasma samples at high [HSA]). Following the same pattern, the apparent stoichiometry of binding (though independent of sample identity and concentration) was higher in equilibrium dialysis (n(app) = 3.2 +/- 0.10) than in fluorescence titration (n(app) 1.9 +/- 0.30). The difference between the two methods could be reconciled by invoking two distinct classes of binding sites (I and II), which had identical (or marginally different) K(d) values, while differing in the magnitude of the fluorescence signal (Deltaf) generated upon ligand binding (Deltaf, PL(I) = Deltaf(I); Deltaf, PL(II) = 0). Further, it was assumed that the state of occupation of class II sites affected the fluorescence efficiency of class I sites, such that Deltaf, PL(I,II) = betaDeltaf(I) (beta = interaction factor). A random binding scheme involving P, PL(I), PL(II), and PL(I,II) was formulated. The model adequately predicted the behavior of the system when monitored through the change in protein fluorescence: Taking K(d) = 25 microM and n(T) = 3, the interaction factor beta was found to be 0.62 +/- 0.10. It was concluded that the correct parameters for the binding of salicylate ion to HSA are K(d) = 25 +/- 2.4 microM and n(T) = 3.2 +/- 0.10, as indicated by equilibrium dialysis of purified HSA. Besides updating information relating to the salicylate binding potential of HSA, this study serves to illustrate a likely complication in the study of protein-ligand interactions by fluorometric methods.     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.