Presence Of Nonidet Research Articles

Phosphorylation of Adult Type Sept5 (CDCrel-1) by Cyclin-dependent Kinase 5 Inhibits Interaction with Syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.

In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors α and β Supports an Active Displacement Model for Coregulator Utilization

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.

Identification and Functional Characterization of a Presqualene Diphosphate Phosphatase

Presqualene diphosphate (PSDP) is a bioactive lipid that rapidly remodels to presqualene monophosphate (PSMP) upon cell activation (Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997) Nature 389, 985-990). Here, we have identified and characterized a phosphatase that converts PSDP to PSMP. Unlike the related polyisoprenyl phosphate farnesyl diphosphate (FDP), PSDP was not a substrate for type 2 lipid phosphate phosphohydrolases. PSDP phosphatase activity was identified in activated human neutrophil (PMN) extracts and partially purified in the presence of Nonidet P-40 with gel filtration and anion exchange chromatography. Peptide sequencing of a candidate phosphatase was consistent with phosphatidic acid phosphatase domain containing 2 (PPAPDC2), an uncharacterized protein that contains a lipid phosphate phosphohydrolase consensus motif. Recombinant PPAPDC2 displayed diphosphate phosphatase activity with a substrate preference for PSDP > FDP > phosphatidic acid. PPAPDC2 activity was independent of Mg(2+) and optimal at pH 7.0 to 8.0. Incubation of [(14)C]FDP with recombinant human squalene synthase led to [(14)C]PSDP and [(14)C]squalene formation, and in the presence of PPAPDC2, [(14)C]PSMP was generated from [(14)C]PSDP. PPAPDC2 mRNA was detected in human PMN, and is widely expressed in human tissues. Together, these findings indicate that PPAPDC2 in human PMN is the first lipid phosphate phosphohydrolase identified for PSDP. Regulation of this activity of the enzyme may have important roles for PMN activation in innate immunity.

Early events in the activation of Fc gamma RIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of Fc gamma RIIA.

The signal transduction mechanisms associated with the ligation of FcgammaRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcgammaRIIA following its cross-linking. The results obtained indicate that cross-linking of FcgammaRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcgammaRIIA in the cells. The stimulated degradation of FcgammaRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcgammaRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by beta-octylglucoside. Sucrose gradient analysis of the distribution of FcgammaRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcgammaRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcgammaRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcgammaRIIA and provide new insights into the early events following FcgammaRIIA cross-linking.

Soluble, Template-Dependent Extracts from Nicotiana benthamiana Plants Infected with Potato Virus X Transcribe both Plus- and Minus-Strand RNA Templates

We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3′ terminal region were not copied, suggesting that elements in the 3′ region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.

Purification and Characterization of a Glucuronyltransferase Involved in the Biosynthesis of the HNK-1 Epitope on Glycoproteins from Rat Brain

The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid, i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group.

DNA determinants and substrate specificities of Escherichia coli MutY.

Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro-8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein.

Regulation of phosphoinositide kinases in T cells. Evidence that phosphatidylinositol 3-kinase is not a substrate for T cell antigen receptor-regulated tyrosine kinases.

A phosphoinositide kinase that can phosphorylate phosphatidylinositol (PtdIns) is present in 4G10 monoclonal antibody (mAb) phosphotyrosine immunoprecipitates isolated from T cells activated via the T cell antigen receptor (TCR).CD3 complex. This PtdIns kinase is not the PtdIns 3-kinase that associates with activated protein tyrosine kinases in fibroblasts, since Western blotting and immunoprecipitation experiments with antibodies specific for the p85 alpha subunit of the PtdIns 3-kinase indicate that this polypeptide is not immunoprecipitated by the 4G10 mAb from TCR.CD3-activated Jurkat cells. Moreover, immunoprecipitated PtdIns 3-kinase isolated from T cells with p85 antibodies is inhibited when PtdIns is presented in Nonidet P-40, whereas the PtdIns kinase activity present in 4G10 mAb phosphotyrosine immunoprecipitates is enhanced in the presence of Nonidet P-40. In vitro kinase assays of PtdIns 3-kinase immunoprecipitated with p85 antibodies from T cells indicate that it associates with a serine kinase that can phosphorylate a p85 polypeptide. However, no protein tyrosine kinase activity capable of tyrosine phosphorylating p85 in vitro associates with p85 alpha immunoprecipitates in quiescent or TCR.CD3-activated T cells. These data suggest that the TCR.CD3 complex does not regulate PtdIns 3-kinase activity by a mechanism that involves protein tyrosine kinases.

A structural polypeptide of the baculovirus Autographa californica nuclear polyhedrosis virus contains O-linked N-acetylglucosamine.

A structural glycopeptide, gp41, derived from the occluded virus of the baculovirus Autographa californica nuclear polyhedrosis virus was characterized. The peptide specifically bound wheat germ agglutinin but was not recognized by a panel of seven other lectins. Reactivity with wheat germ agglutinin was eliminated by treatment of gp41 with beta-N-acetylglucosaminidase, indicating that N-acetylglucosamine (GlcNAc) was present as terminal residues. gp41 was efficiently galactosylated by galactosyltransferase only in the presence of Nonidet P-40, suggesting that GlcNAc residues are not exposed on the surface of the virion. Metabolic labelling of gp41 with [3H]GlcNAc occurred in the presence of tunicamycin. The carbohydrate was released by alkaline borohydride treatment and comigrated with N-acetylglucosaminitol in descending paper chromatography. The data indicate that gp41 contains single residues of GlcNAc O glycosidically linked to the polypeptide chain. Evidence suggesting that gp41 is located in the region between the envelope membrane and the capsid (defined here as the tegument) of the occluded virus is also presented.

Electric properties of photoaffinity-labelled pancreatic A-subtype cholecystokinin

Although the isoelectric point of a protein is very important, electric focusing of intrinsic membrane proteins in polyacrylamide or agarose gels often fails. The recently introduced Bio-Rad Rotofor cell allowed isoelectric focusig of such a protein, cholecystokinin (CCK) receptor. Both the isoelectric point and the molecular weight (Mr) of pacreatic CCK receptor were determined. For this purpose, membrane CCK receptor was photoaffinity labelled by a cleavable agonist probe, subsequently prepurified on immobilized wheat germ agglutinin and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectrofocusig in solution in the presence of Nonidet P-40. CCK receptor was identified at Mr 85 000–100 000, whereas its deglycosylated product was shifted to M4 42 000. Further, the isoelectric poits of the glycosylated and deglycosylated forms of CCK receptor were pH 4.8 and 4.3, respectively. A knowledge of the isolelectric point should help in characterizing better CCK receptor heterogeneity and/or in purifying CCK receptor proteins.

Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse.

We have demonstrated previously that spermatogenic cells in the mouse testis have high levels of fucosyltransferase activity. Furthermore, a significant portion of this activity has been localized to the cell surface (Millette et al.: Cell Biology of the Testis and Epididymis, 1987). Differential expression of fucosyltransferases and their function as ecto-enzymes may be important in the processes of sperm maturation and fertilization in mammals. Accordingly, here we report the activity levels of fucosyltransferase (FT) in spermatozoa isolated from the mouse caput and cauda epididymides. Calculated on a per cell basis, spermatozoa from the caput epididymis have significantly more FT activity than do spermatozoa from the cauda epididymis (18.07 +/- 2.2 pmol/million cells compared with 2.8 +/- 0.09 pmol/million cells). Furthermore, caput sperm exhibit a more significant increase in FT activity when assayed in the presence of Nonidet P-40. Calculated on the basis of cell surface area, however, FT activity remains constant on the head portion of spermatozoa isolated from all portions of the male reproductive tract and from capacitated spermatozoa. Measurements of FT activity in extracts of isolated sperm tails from cells at different stages of maturation indicate a greatly diminished activity in tails from sperm in the cauda epididymis. The total sperm surface area is composed predominantly of the plasma membrane surrounding the flagellar apparatus. Therefore, our data demonstrate that FT activity is retained selectively on the different topological regions of sperm, with losses during sperm maturation in the epididymis being restricted to the tail segment. Maintenance of high levels of FT activity of the plasma membranes of the mouse sperm head raise the possibility that FT is indeed involved in some aspects of sperm-egg recognition.

Approach to stationary two‐dimensional pattern: Influence of focusing time and Immobiline/carrier ampholytes concentrations

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Görg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.

Activation of dolichyl-phospho-mannose synthase by phospholipids.

Dolichyl-phospho-mannose synthase, or GDPmannose:dolichyl-phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl beta-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207-218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 115-1119]. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The Km for GDP-mannose was found to be 0.69 microM and the apparent Km for dolichyl-P was 0.3 microM. GMP, GDP, and GTP inhibited mannosyl transfer 50% at concentrations of 16 microM, 1.3 microM and 3 microM respectively.

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with nonidet P-40

The extraction of [ 3H]estradiol- and [ 3H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [ 3H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [ 3H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [ 3H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S. The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.

Artefacts associated with Nonidet P‐40 upon sodium dodecyl sulphate polyacrylamide gel electrophoresis of protein

AbstractOne‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of protein in the presence of Nonidet P–40 (NP–40) induces vertical streaking which breaks horizontal bands of protein to produce a “multiple spot” effect. The same effect can be induced upon high resolution two‐dimensional electrophoresis if the NP–40 concentration of the first‐dimension isoelectric focusing (IEF) gels is increased to 4 % w/v and the gels electrophoresed (SDS‐PAGE) without SDS‐equilibration. The relevance of this phenomenon as regards interpretation of two‐dimensional electrophoretic patterns is discussed.

Glycoproteins of Sendai virus (HVJ) have a critical ratio for fusion between virus envelopes and cell membranes

The biological activity of two glycoproteins, hemagglutinin and neuraminidase (HN) and fusion (F) proteins, of Sendai virus (HVJ) were studied using purified proteins. The proteins were purified by chromatography on DEAE and CM cellulose in the presence of Nonidet P-40 (NP40). The glycoproteins were reconstituted at various ratios of F to HN into lipid vesicles containing fragment A of diphtheria toxin. The association of HN and F proteins with the vesicles was confirmed by electron microscopy and sucrose density gradient centrifugation. The cytotoxic activity of vesicles containing fragment A on fusion with L cells was determined by measuring colony formation of the cells. It was found that for maximum cytotoxic activity of the vesicles, there was an optimal ratio of F to HN of two. This suggests that HN is not merely the initial binding site to the cell surface, and that interactions between HN and F proteins on the virus surface may be important for the biological activities of these proteins on the cells.

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.

Human T gamma globin chain is a variant of A gamma chain (A gamma Sardinia).

Isoelectric focusing, cellulose acetate electrophoresis, and carboxymethylcellulose chromatography in the presence of Nonidet P-40 allow the separation of pure gamma chains into two fractions. Amino acid analysis of their cyanogen bromide fragment 3 (gamma CB3) identifies these fractions as the separated G gamma (Gly-136) and A gamma (Ala-136) globin chains. Fingerprint and amino acid analyses of the gamma Tp9 tryptic peptide from the purified A gamma and G gamma fractions from two different patients demonstrate that the commonly occurring gamma Sardinia variant (gamma 75 isoleucine leads to threonine), also known as T gamma chain, has alanine in position 136. From this analysis we suggest that the T gamma gene is an allele of the A gamma locus (A gamma Sardinia) rather than a third gamma locus.

Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking

To examine the protein proximity and subunit organization of type C retroviruses, preparations of AKR murine leukemia virus were treated with bifunctional cross-linking reagents and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cross-linked components obtained were characterized by immunoprecipitation with monospecific antisera against purified viral proteins, followed by SDS-PAGE analysis both before and after cleavage of the cross-links. With these procedures, complexes of both viral envelope and core components were identified. The major envelope subunit obtained was a large (apparent molecular weight of 450,000 to 500,000), glycosylated complex, composed of four to six gp70-p15(E) subunits. This complex was detected over a 100-fold range of cross-linker concentration and thus seems to represent a particularly stable viral substructure. The cross-linked complexes of the core proteins consisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic structural unit of the viral core. When virion preparations, which had previously been disrupted with the nonionic detergent Nonidet P-40, were cross-linked, the envelope complex was still observed, indicating that this structure is stable in the presence of Nonidet P-40. A similar envelope structure was observed for feline leukemia virus, suggesting that such a complex may be a conserved feature of oncornavirus structure.

G γ and A γ globin chains separation and quantitation by isoelectric focusing

Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the G γ and A γ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to G γ and the band at pI 6.85 corresponds to the A γ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only A γ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative G γ A γ synthetic ratios are similar to the relative amounts of G γ and A γ chains accumulated in the erythrocytes, suggesting that the activities for the G γ and A γ mRNAs decay at roughly similar rates.