Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a).

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.

Substituted poly(methyl vinyl ether-alt-maleic anhydride) for the release control and targeting of methotrexate.

Poly(methyl vinyl ether-alt-maleic anhydride) substituted with cholamine (CA), aminoethylcholamine (AECA), or aminooctylcholamine (AOCA) at different substitution degrees, were used for methotrexate (MTX) complexation. The solid complexes, isolated by precipitation from the preparative mixture, showed lower fractional releases at pH 7.4 than at 5.5. This was ascribed to the establishment of ionic interactions between the ionized carboxyls of both the polymer and the drug and the quaternary ammonium groups of the substituents (CA, AECA, AOCA) inducing polymer self-aggregation and thus complex stabilization. The fractional release in pH 7.4 decreases with the increase in the substitution degree until a minimum characteristic for each substituent analyzed is reached and then rises with the increase in substitution degree. The minimum release at pH 7.4 was observed in the presence of AECA at the degree of substitution corresponding to 0.35 mole of substituent per mole of dimer (methyl vinyl ethermaleic anhydride). None of the substituted polymers studied had any haemolytic effect, indicating good biocompatibility.

Chemical modification of cationic groups in the polypeptide cardiac stimulant anthopleurin-A.

Chemical modification studies have been carried out on the sea anemone polypeptide anthopleurin-A in order to clarify the role of Arg-14 in its cardiac stimulatory activity. Reaction with 1,2-cyclohexanedione at 37 degrees C produced a range of protein products, including some with amino group modifications. These side-reactions were eliminated by prior citraconylation of the amino groups, which, following reaction with cyclohexanedione, could be reversed under conditions which preserved the cyclohexanedione adduct. Citraconylation of the three amino groups, one from the N-terminus and two from Lys-37 and Lys-48, destroyed the cardiac stimulatory activity of the molecule, but this was fully recoverable upon reversal of this reaction. It appears that one or more of the amino groups is essential for activity. Anthopleurin-A contains only one arginine residue, and this was confirmed as the site of modification by cyclohexanedione by showing that the product was refractory to proteolysis by trypsin, which normally cleaves the molecule at this residue. The positive inotropic activity of the cyclohexanedione adduct on isolated guinea-pig atria was identical to that of unmodified anthopleurin-A, indicating that the side-chain of Arg-14 is not required for cardiotonic activity.

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement.

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.

Schistosoma mansoni: chemical stabilization of cercariae by aldehydes.

Chemically stabilized cercariae of Schistosoma mansoni have been developed by inactivating surface glycoproteins which are essential for their survival. The inactivation was achieved by reaction with 0.01-0.1% glutaraldehyde, 0.1-1% formaldehyde, and 0.37-3.7 microliters citraconic anhydride. The cercariae lost their viability but retained the ability to exclude trypan blue for up to 2 years in a manner similar to live cercariae and in contrast to cercariae killed by other means, which took up the dye immediately. The chemically stabilized cercariae reacted with polyclonal and monoclonal antischistosome antibodies in an indirect immunofluorescence assay for up to 2 years, indicating the retention and preservation of surface antigens. Chemically stabilized cercariae revealed the presence of antischistosome antibodies as early as 1 week after infection when used for immunodiagnosis of mouse and rat infections. The presence of Fc receptors for human IgG on the stabilized cercariae interfered in their use as an immunodiagnostic reagent of human schistosomiasis. The stabilized cercariae were also used to screen cultures for monoclonal antischistosome antibodies. Preliminary results indicated that immunization of mice with glutaraldehyde stabilized cercariae imparted protective immunity to mice.

Phosphorylation site of eukaryotic initiation factor 4E.

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.

Effects of amino group modification in discoidal apolipoprotein A-I-egg phosphatidylcholine-cholesterol complexes on their reactions with lecithin:cholesterol acyltransferase.

Discoidal complexes of human apolipoprotein A-I-egg phosphatidylcholine-cholesterol were prepared by the sodium cholate dialysis procedure and were reacted to varying extents with the amino group reagents citraconic anhydride, diketene, and formaldehyde in the presence of sodium borohydride. Modification of positive lysine residues with negative or neutral groups (citraconic anhydride and diketene, respectively) resulted, for extensively reacted complexes (90%), in structural alterations and in a marked decrease in reactivity with purified human lecithin:cholesterol acyltransferase. The structural and kinetic effects were partially reversible by removal of the modifying groups or by increased ionic strength. Similar extents of modification (84%) with retention of positive charge and introduction of two methyl groups (reductive methylation) had no effect on the structure or the reactivity of the complexes. These results, together with kinetic data at variable complex concentrations or at variable temperatures, indicate that specific lysine residues of apolipoprotein A-I are not involved in the lecithin:cholesterol acyltransferase activation process; instead, charge interactions and structural changes are responsible for the observed decrease in activating capacity. In terms of kinetic parameters, intrinsic K*m values and probably enzyme-substrate particle dissociation constants are affected, but the activation energies remain the same upon chemical modification.

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei.

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.

The action of thrombin on modified fibrinogen.

The modification of canine fibrinogen with citraconic anhydride modified the epsilon-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

The cell surface of Plasmodium gallinaceum sporozoites: microelectrophoretic and lectin-binding characteristics.

The cell surface properties of Plasmodium gallinaceum sporozoites have been investigated by means of microelectrophoretic and lectin-binding studies. Their electrophoretic mobility has been measured as a function of pH, the results suggesting qualitative and quantitative differences in the surface ionogenic groups between sporozoites from mature oocysts and those from salivary glands. Reaction of sporozoites with citraconic anhydride produced a small but significant increase in mobility, whereas 5,5-dithio-bis-(2-nitrobenzoic) acid had no effect on mobility; thus there appear to be amino groups but not -SH groups at the surface of sporozoites. Treatment of sporozoites with trypsin considerably reduced their mobility and suggests that a significant proportion of the cell surface charge is associated with protein. Incubation with neuraminidase, however, had no effect on sporozoite mobility and indicates that sialic acid residues, responsible for much of the negative charge associated with mammalian cells, are probably not present on the cell surface of sporozoites. Evidence for the presence of carbohydrates on the cell surface membrane of sporozoites was sought using fluorescein isothiocyanate-Concanavalin A. Results demonstrated that ligands similar to alpha-D-glucose and alpha-D-mannose are not present in an exposed or reactive form on the cell surface membrane of P. gallinaceum sporozoites.

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.

[Use of N-benzoyl-phenylalanyl-valyl-arginine p-nitroanilide and hirudin for the detection of enzymatic activity under prothrombin acylation by citraconic anhydride]. / Ispol'zovanie N-nitroanilida N-benzonl-fenilalanil-valil-arginina i girudina dlia vyiavleniia fermentativnoi aktivnosti pri atsilirovanii protrombina tsitrakonovym angidridom.

Acylation of bovine prothrombin by cytraconic anhydride results in the formation of the thrombin active site, which provides the degradation of highly specific thrombin substrate, N-bensoyl-Phe-Val-Arg p-nitroanilide. This reaction is inhibited with hirudin, highly specific thrombin inhibitor.

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.

Some effects of positively charged surface groups on cell aggregation.

A study was made of the effects of 2,3-dimethylmaleic anhydride (DMA), a reagent removing positive charges, on the aggregation and surface charge of embryonic chick neural retina cells. Neural retina cells, recovered from the dissociation procedure, were cultured on a gyratory shaker and the aggregate dimaeters formed in the presence of DMA or DMA-serum dialysate, following DMA-pretreatment, or in appropriate control cultures measured. The electrophoretic mobilities of similarly treated cells were also determined. In addition, cellulose acetate electrophoresis was carried out on samples of serum containing DMA, and the incorporation of 14C-amino acids into DMA-treated cells studied. Aggregates formed in the presence of DMA, or following DMA-pretreatment, were significantly smaller than aggregates from control cultures. The electrophoretic mobility of DMA-treated cells was significantly increased in serum-containing medium, but not serum-free Hanks' solution. At 24 h after removal of DMA-containing medium, the mobilities of pretreated cells were similar to those of controls. The electrophoretic pattern of DMA-treated serum was changed only with concentrations of DMA many times that affecting cell aggregation or mobility. DMA-serum dialysate did not significanlty reduce aggregate size. The incorporation of 14C-amino acids in DMA-treated cells and the structure of aggregates were unchanged from controls. It is concluded that positively charged consituents of the cell periphery play a demonstrable, but not limiting, role in cell aggregation, while a minor role for positive charges on serum protein cannot be totally excluded.