Demonstration of multilineage chimerism in a nonhuman primate concordant xenograft model.

Prior studies from our laboratory have demonstrated that a nonmyeloablative conditioning regimen can induce transient mixed chimerism and renal allograft tolerance between MHC disparate cynomolgus monkeys. We have also shown that this preparative regimen can be extended to a concordant baboon to cynomolgus xenograft model by adding, to the post transplant protocol, therapy designed to prevent antibody production. Here we examine the use of brequinar (BQR) for this purpose and the efficacy of two new reagents developed to demonstrate the establishment of chimerism in the xenograft recipients. The cynomolgus recipients were conditioned with WBI (300 cGy), TI (700 cGy), ATG, cyclosporine, and brequinar sodium. To detect engraftment of the donor marrow, we prepared a polyclonal cynomolgus anti-baboon antibody (CABA) and a monoclonal antibody (215.1), which distinguish baboon and cynomolgus lymphocytes and granulocytes. We employed flow cytometry analysis to detect multilineage chimerism in the xenograft recipients. Five of the six recipients monitored using our new reagents (CABA and 215.1) developed detectable chimerism and only one of these animals lost its kidney to rejection. However, other complications have not permitted assessment of long-term outcome. The features of the multilineage chimerism included the detection of donor granulocytes (1.8-77.4%) and lymphocytes (2.4-22.2%) for 9 to 37 days. Our new reagents permit the detection of multilineage mixed chimerism, which may be a predictor of xenograft tolerance. We also conclude that brequinar may be effective in preventing antibody formation, but because of its toxicity, it is probably not the drug of choice for extension of the mixed chimerism protocol to concordant xenografts.

DNA binding domain of Escherichia coli DNA polymerase I: identification of arginine-841 as an essential residue.

To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), we have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with [7-14C]PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. An appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that the two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modification, which implicates this residue in the DNA binding function of pol I.

Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex.

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.

High performance liquid chromatographic properties of peptides and proteins on a dihydroxyalkyl bonded silica stationary phase.

The chromatographic behavior of biologically relevant peptides and proteins in the molecular weight range between 200 and 200,000 dalton units were studied on a size exclusion matrix column consisting of an aqueous compatible dihydroxyalkyl bonded silica support. The mechanism of separation appears to be dependent on hydrodynamic radius, hydrophobic and ionic interactions. Support for this contention is based on the chromatographic properties of these peptides and proteins at different mobile phase ionic strengths and pH, oxidation state of amino acid residues and total hydrophobicity of the peptide or protein. This column is also capable of separating native angiotensin-I from its iodinated congener. Recoveries of proteins and peptides from this column ranged between 70-100%. Unlike typical reverse phase separations, this modified silica chromatographic media allows for an alternative technique employing aqueous eluents for rapid separation/isolation and purification of peptides and proteins from natural or synthetic sources.

Mechanism of o-phenanthroline mediated inhibition of E. coli DNA polymerase I : formation of template-primer-metal-phenanthroline complexes with resultant loss of catalytic activity.

Inhibition of E. coli DNA polymerase I activity by 1,10 phenanthroline in the absence of reducing agents requires a high concentration of inhibitor (1-10 mM) depending upon the template primer used to direct the synthesis. We find that o-phenanthroline, unlike its non-chelating analogue, forms a divalent cation mediated complex with template-primers. Enzyme bound to such complexes is unable to catalyse either polymerization or nuclease functions.

Biochemistry of terminal deoxynucleotidyltransferase: characterization and properties of photoaffinity labeling with 8-azidoadenosine 5'-triphosphate.

We have found that 8-azidoadenosine 5'-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase with respect to substrate deoxynucleoside triphosphates. A detailed characterization of the inhibitory effect of 8-azido-ATP revealed that its mechanism of inhibition is identical with that reported for ATP [Modak, M. J. (1978) Biochemistry 17, 3116-3120]. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to terminal deoxynucleotidyltransferase. No significant incorporation of prephotolyzed azido-ATP or unsubstituted ATP into enzyme protein is noted when complexes of these nucleotides with enzyme were exposed to identical photoactivation conditions. The majority of incorporated analogue was associated with the 26 000-dalton subunit of terminal deoxynucleotidyltransferase. Incorporation of azido-ATP was further found to be absolutely dependent on the presence of a divalent cation. All four deoxyribonucleoside triphosphates as well as ATP and guanosine 5'-triphosphate were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analogue to terminal deoxynucleotidyltransferase (TDT). In addition, substrate binding site directed inhibitors, pyrophosphate and pyridoxal 5'-phosphate, effectively blocked the incorporation of azido-ATP into enzyme protein, while several other inhibitors of TDT catalysis, namely, ethylenediaminetetraacetic acid, alpha, alpha'-dipyridyl, 1,10-phenanthroline, p-(chloromercuri)benzoate, Rose Bengal, and the presence of 0.5 M KCl, influenced the cross-linking reaction to varying degrees. A tryptic peptide analysis of the azido-ATP-labeled 26K subunit of TDT revealed that the majority of the incorporated photoaffinity analogue was present in two peptides.

The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function.

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.

Studies on proteinases from Calotropis gigantea latex. I. Purification and some properties of two proteinases containing carbohydrate.

Two proteinase containing carbohydrate, called calotropain-FI and calotropain-FII, were purified from Calotropis gigantea latex by CM-Sephadex C-50 chromatography. Both calotropain-FI and FII were found to be homogeneous by rechromatography on CM-Sephadex C-50, gel filtration on Sephadex G-100, electrophoresis on polyacrylamide gel and by N-terminal amino acid analysis. Some properties of these enzymes are reported.

Studies on proteinases from Calotropis gigantea latex. II. Physico-chemichal properties of calotropain-FI and FII.

The molecular weights of purified calotropain-FI and FII were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by filtration of Sephadex G-100. Activation of calotropain-FI and FII by different sulfhydryl activators was studied. The results obtained from inhibition studies by various enzyme-modifying reagents suggest the possible role of cysteine and histidine residues in the active site of both the enzymes. The free and total sulfhydryl contents of both the enzymes were determined by the use of 5-5'-dithio-bis-2-nitrobenzoic acid. Total amino acid compositions of both the enzymes were also determined. A comparative study of the esterase, amidase, milk-clotting and caseinolytic activities of calotropain-FI and FII are also presented.

Separation of chymopapain from papaya latex (Carica papaya) on amberlite IR-120 (Hg2+).

The quantitative separation of chymopapain from papaya latex has been carried out by chromatography on Amberlite IR-120 (Hg2+). The product obtained was further studied to determine its homogeneity.