The effect of neuraminidase on genetic variants of alpha anitrypsin.

Sera of Pi types M, F, S, Z, IM, FM, MS, and MZ were incubated with neuraminidase and the reaction products followed by electrophoresis. The alpha1 antitrypsin components showed a series of changes in mobility as sialic residues were removed. Removal of sialic acid was confirmed by chemical assay. Results of studies with two different electrophoretic systems suggested that the Z type alpha1 antitrypsin has less sialic acid than the M, F, and S types. There was no evidence that other genetic variants have a reduced sialic acid content. The two major bands of alpha1 antitrypsin seen in certain electrophoretic systems may reflect a difference of one sialic acid residue. It is proposed that the Z protein lacks a carbohydrate chain with two terminal sialic acid residues. This carbohydrate deficiency results in lack of secretion of type Z alpha1 antitrypsin from the endoplasmic reticulum, perhaps because of binding to sites specific for the incomplete glycoprotein or because of aggregation of the Z asialo protein. A carbohydrate chain could be prevented from attaching to the Z type either because of a conformational change or because of the replacement of a carbohydrate-binding asparagine residue in the Z protein.     

Glycosylation study of the major genetic variants of human alpha1-acid glycoprotein and of their pharmacokinetics in the rat

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants, the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. AAG is also an extensively glycosylated protein which possesses five N-linked glycans exhibiting substantial heterogeneity in their structures. The first objective of this study was to investigate the glycosylation of the two major gene products of AAG, i.e. the A variant and a mixture of the F1 and S variants (F1*S). To this end, we combined a chromatographic method for the fractionation of the AAG variants with a lectin-binding assay to characterise the glycosylation of purified glycoproteins. Secondly, because the oligosaccharides can influence the disposition of AAG, a kinetic study of the AAG variants was carried out in the rat. After intravenous administration of whole human AAG, the separation and quantification of the AAG variants in plasma was performed by application of specific methods by isoelectric focusing and immunonephelometry. The binding studies carried out on a panel of lectins showed significant differences in the lectin-binding characteristics of the separated F1*S and A variants, accounting for differences in the degree of branching of their glycan chains and substitution with sialic acid and fucose. The plasma concentration-time profiles of the F1*S and A variants were biphasic, and only small differences were observed between the variants for their initial and terminal half-lives, clearance and distribution volume. This indicates that the structural differences between the two AAG gene products do not affect their pharmacokinetics in the rat. Specific drug transport roles have been previously demonstrated for the F1*S and A variants, calling for further investigations into their effects on the disposition of drugs they bind in plasma. The present study shows that such investigations are possible without being complicated by kinetic differences between these variants.     

Separation of both the Bbeta- and the gamma-polypeptide chains of human fibrinogen into two main types which differ in sialic acid content

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen have each been resolved into two major species: gammaL and gammaR and BbetaL and BbetaR. These molecular variants, separable on CM-cellulose, differ from each other in sialic acid content: approximately 2 residues of sialic acid per molecule of polypeptide chain for the L species to 1 residue of sialic acid per molecule for the R species. The two types of each polypeptide are demonstrable in preparations of fibrinogen from single donors as well as in pooled fibrinogen. The L and R forms of the gamma chains or the Bbeta chains do not differ in their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting that they are similar in molecular weight. They are also indistinguishable in polyacrylamide gels in the presence of urea at pH 2.7. Maps of ninhydrin-positive tryptic peptides of the L and R forms of the gamma chain displayed differences within a small group of peptides which have been shown to contain the sialic acid residues present in the gamma-polypeptides. No differences between the peptide maps of BbetaL and BbetaR chains were obvious. A larger ratio of L/R in the gamma and Bbeta chains of dysfibrinogenemia fibrinogen "Zürich II" than in those of normal fibrinogen explains the higher content of sialic acid measured in the native Zürich II fibrinogen molecule.     

Structural studies on individual components of bovine transferrin

The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ;maps', although differences in composition and peptide ;maps' occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain differences in electrophoretic mobility between bands of the same variant which are not produced by differing sialic acid content.     

Involvement of conserved histidine, lysine and tyrosine residues in the mechanism of DNA cleavage by the caspase-3 activated DNase CAD

The caspase-activated DNase (CAD) is involved in DNA degradation during apoptosis. Chemical modification of murine CAD with the lysine-specific reagent 2,4,6-trinitrobenzenesulphonic acid and the tyrosine-specific reagent N-acetylimidazole leads to inactivation of the nuclease, indicating that lysine and tyrosine residues are important for DNA cleavage by this enzyme. The presence of DNA or the inhibitor ICAD-L protects the enzyme from modification. Amino acid substitution in murine CAD of lysines and tyrosines conserved in CADs from five different species leads to variants with little if any catalytic activity, but unaltered DNA binding (K155Q, K301Q, K310Q, Y247F), with the exception of Y170F, which retains wild-type activity. Similarly, as observed for the previously characterised H242N, H263N, H308N and H313N variants, the newly introduced His→Asp/Glu or Arg exchanges lead to variants with <1% of wild-type activity, with two exceptions: H313R shows wild-type activity, and H308D at pH 5.0 exhibits ~5% of wild-type activity at this pH. Y170F and H313R produce a specific pattern of fragments, different from wild-type CAD, which degrades DNA non-specifically. The recombinant nuclease variants produced in Escherichia coli were tested for their ability to form nucleolytically active oligomers. They did not show any significant deviation from the wild-type enzyme. Based on these and published data possible roles of the amino acid residues under investigation are discussed.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.     

Partial characterization of the sialic acid-free forms of α1-acid glycoprotein from human plasma

Some of the properties of sialic acid-free α₁-acid glycoprotein prepared by mild acid hydrolysis (pH1·6 at 80° for 1hr.) were compared with those of neuraminidasetreated α₁-acid glycoprotein. Chemically, the former contained less fucose (15%) and amide (2%) residues. Physicochemically, it had undergone certain changes primarily pertaining to the secondary structure, so that the specific optical rotation was more negative than that of the latter. A further expression of this change is probably the difference in the pH range of the resolution into two bands on electrophoresis. The resolution of the glycoprotein prepared by mild acid hydrolysis seems to be extended to more acidic pH values both by starch-gel and free moving-boundary electrophoresis. On ultracentrifugation both preparations appeared homogeneous and sedimented with a rate of 3s. Removal of sialyl residues at different pH values, in the range 1–7, showed that 2moles of sialic acid/mole of protein are very strongly bound. The two variants of α₁-acid glycoprotein were isolated from pooled sialic acid-free α₁-acid glycoprotein by preparative starch-gel electrophoresis and from selected blood of normal adults by fractionation by solubility and chromatography. Ultracentrifugal and starch-gel electrophoretic analyses at pH5, with incubation times of 1 or 24hr., demonstrated that no dissociation–association equilibrium (constant sedimentation coefficient and molecular weight) or isomerization (constant apparent electrophoretic mobilities) exist between the two variants. Therefore these variants are not sub-units of native α₁-acid glycoprotein but represent modifications of naturally occurring proteins. Further, it was shown that the difference in the electrophoretic mobilities between the two variants was not due to any difference in amide content. Immunochemically, the two variants share the same determinants.     

Kinetic and mechanistic studies of penicillin-binding protein 2x from Streptococcus pneumoniae.

High molecular weight penicillin-binding proteins (PBPs) are bifunctional enzymes that build bacterial cell walls from the glycopeptide lipid II [GlcNAc-MurNAc(L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala)-pyrophosphate-undecaprenol] by a process involving disaccharide polymerization and peptide cross-linking. The latter reaction involves acyl-transfer chemistry in which the penultimate (D)Ala first acylates the active-site serine, with release of the terminal (D)Ala, and is then transferred to the epsilon-amine of a Lys on a neighboring pentapeptide chain. These enzymes also catalyze hydrolysis of specific thioester substrates and acylation by beta-lactam antibiotics. In this paper, we explore these latter two reactions and report mechanistic experiments on the reaction of Streptococcus pneumoniae PBP 2x with N-benzoyl-(D)Ala-thioacetic acid [Bz-(D)Ala-(S)Gly] and penicillin G. For these experiments, we used PBP 2x, a soluble form of PBP 2x in which the transmembrane domain was deleted. The following results are significant: (1) pH dependencies for acylation of PBP 2x by penicillin G and Bz-(D)Ala-(S)Gly are identical, suggesting that the same ionizable residues are involved in both reactions and that these residues play the same catalytic role in the two processes. On the basis of these results, we propose a mechanistic model that is also consistent with recently published structural data [Gordon, E., et al. (2000) J. Mol. Biol. 299, 477-485]. (2) Pre-steady-state experiments for the PBP 2x-catalyzed hydrolysis of Bz-(D)Ala-(S)Gly at pH 6.5 indicate that k(c) is principally rate-limited by acylation with some contribution from deacylation. The contribution of these steps to rate limitation is pH-dependent, with acylation entirely rate-limiting at pH values less than 5.5 and deacylation principally rate-limiting above pH 8.5. (3) Results of solvent isotope effect and proton inventory experiments for acylation suggest a complex process that is at least partially rate-limited by chemistry with some involvement of changes in solvation and/or enzyme conformation. (4) Analysis of activation parameters suggests that during the acylation of PBP 2x by penicillin G the inherent chemical stability of penicillin's amide bond, as manifested in the enthalpy of activation, is offset by a favorable entropy term that reflects penicillin's rotationally constrained bicyclic system, which presumably allows a less energetically demanding entry into the transition state for acylation.     

Genetic studies of low-abundance human plasma proteins. XIII. Population genetics of C1R complement subcomponent and description of new variants

Isoelectric focusing and immunoblotting reveals considerable biochemical and genetic variation in the C1R subcomponent of the first complement component. The nature of the intraindividual biochemical variation can be explained by differences in sialic acid content because after digestion with neuraminidase the terminal sialic acids are removed to yield a single major band corresponding to the C1R polypeptide. Plasma samples from a large number of different ethnic groups, consisting of U.S. whites, U.S. blacks, Nigerian blacks, and Inuit, Aleut, and Amerindian populations from the Western Hemisphere have revealed genetically determined charge variation with heterozygous phenotypes consisting of two major asialo bands, indicating that the underlying variation is not due to variation in sialic acid content. Two previously reported common alleles, C1R*1 and CIR*2, have been observed in all studied populations, the notable exception being the Dogrib Indian population, which is devoid of the C1R*2 allele. Several new alleles--designated C1R*3, C1R*4, C1R*5, C1R*6, and C1R*7-have been observed, with variable frequencies ranging from the occurrence in a single individual and related family members to the polymorphic occurrence of certain alleles in several populations. Of these new alleles, the C1R*5 is of considerable interest in population and anthropological genetics studies. The C1R*5 allele is widely distributed, at a frequency of .03 to .17, in all of the North American aboriginal populations screened. This allele is not present in U.S. whites but is present at a polymorphic frequency in U.S. and Nigerian blacks.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Asymmetric gramicidin channels: heterodimeric channels with a single F6Val1 residue.

Substitution of Val1 by 4,4,4,4',4',4'-F6Val in [Val1]gramicidin A ([Val1]gA) produces channels in which the effects of amino acid replacements on dimer stability and ion permeation are nonadditive. If only one Val1 (in a symmetric [Val1]gA channel) is substituted by F6Val, the resulting heterodimeric channels are destabilized relative to both homodimeric parent channels and the single-channel conductance of the heterodimeric channels is reduced relative to the parent channels (Russell, E. W. B., L. B. Weiss, F. I. Navetta, R. E. Koeppe II, and O. S. Andersen. 1986. Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position #1 in gramicidin A. Biophys. J. 49:673; Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence. J. Mol. Biol. 211:221-234). To understand the basis for this destabilization, we have examined further the characteristics of [F6Val1]/[Xxx1]gA heterodimers, where Xxx = Gly, Val, and Ala. These heterodimeric channels show rapid current transitions between (at least) two current levels and display asymmetric i-V characteristics. The orientation of the heterodimers relative to the applied potential was determined by asymmetric addition of the gramicidin analogs, one to each side of a preformed bilayer. The current transitions are most clearly illustrated for [F6Val1]/[Gly1]gA heterodimers, which possess two finite and well defined current levels. Based on the existence of these two conductance states and the analysis of duration and interval distributions, we conclude that the transitions between the two current levels correspond to conformational transitions in "stable" heterodimers. In the case of [F6Val1]/[Val1]gA and [F6Val1]/[Ala1]gA heterodimers, the low-conductance state is indistinguishable from zero. The two (or more) conductance states presumably correspond to different orientations of the dipolar F6Val1 side chain. The distribution between the high- and the low-conductance states varies as a function of potential in [F6Val1]/[Gly1]gA channels. These characteristics cause the [F6Val1]/nonpolar (Val, Ala, Gly)gA hybrid channels to serve as a "simple" model for understanding gating transitions in membrane-spanning channels.     

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

Comparison of bovine serum transferrin A and D2. I. Amino acid residue differences

A comparison is made of single components of the homozygous variants A and D2 of bovine serum transferrin by tryptic, chymotryptic and cyanogen bromide digestion. It is concluded that there are three substitutions A:D2-Glu:Asp, Lys:Arg and Asp:Gly. In the light of the recent work of Brock et al. (1980) it is concluded that all three substitutions occur in the C-terminal sequence of the chain. By homology with the sequence of human serum transferrin (MacGillivray et al., 1982) the Lys:Arg and Asp:Gly substitutions probably occur at residues 527 and 446, respectively, from the N-terminus. The Asp:Gly substitution is considered more likely than our earlier conclusion (Maeda, McKenzie & Shaw, 1977) that there is a deletion in the chain of D2 (A:D2, Asp:--). The location of the Glu:Asp substitution is not known.     

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.     

Expression of Sulfated Gangliosides in the Central Nervous System

Abstract: Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [³⁵S]sulfate. Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14-day-old rat brain with [³⁵S]sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.     

Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly > Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.     

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).