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Biomedical subjects

R A Kahn

Publications and source records attributed to R A Kahn.

At least 109 records · Page 6Linked to original sources
PubMed1989

Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor.

The ADP-ribosylation factor (ARF) is a member of the small molecular weight GTP-binding protein family and serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory regulatory subunit (Gs) of adenylate cyclase. Bovine Arf1 has been expressed at high levels and purified from bacteria. The recombinant Arf1 was compared with purified bovine brain Arf and shown to be nearly identical with respect to immunoblotting, guanine nucleotide binding, GTP hydrolysis, and cholera toxin cofactor activities. The only known chemical difference between the recombinant and brain proteins is the lack of myristic acid at the amino terminus of the expressed protein. The preparation of nucleotide-free Arf1 has allowed a more accurate determination of the binding constants for guanine nucleotides and revealed a significantly higher affinity for GDP than was previously determined. The effect of magnesium ions on nucleotide affinities was also determined and found to be quite different for the different guanine nucleotides. We have shown that GDP binds to the protein in the absence of magnesium, while GTP or guanosine 5'-O-(thiotriphosphate) can only bind to Arf1 in the presence of nanomolar (or higher) levels of the free metal. This characterization of the nucleotide binding and the ability to produce large amounts of a single species of ARF with full retention of a range of activities should greatly facilitate subsequent studies on the structure and function of ARF.

ADP-Ribosylation Factor 1↗
PubMed1988

Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase.

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase. Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced. Approximately 25-30% of the protein was sequenced in this manner. Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins. Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF. Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man. No ARF immunoreactivity was observed when Escherichia coli proteins were tested. Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant. Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein. ARF is shown to be myristylated at the amino terminus. The potential role of myristylation in cellular localization is discussed.

ADP-Ribosylation Factors↗
PubMed1988

Sequences of the bovine and yeast ADP-ribosylation factor and comparison to other GTP-binding proteins.

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein that serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs). An oligonucleotide probe based on the partial amino acid sequence was used to clone ARF from a bovine adrenal chromaffin cDNA library. The yeast (Saccharomyces cerevisiae) ARF gene was then cloned from a YCp50 genomic library by cross-species hybridization by using the coding region of the bovine gene. RNA gel blots of poly(A)+ RNA indicate that only one ARF message size (900 and 2000 base pairs) is present in yeast and cows, respectively. Comparison of the cDNA-derived amino acid sequences of ARF to other GTP-binding proteins reveals a structural relationship between ARF and the ras family of proteins. A slightly better structural relationship is detected when ARF is compared to the alpha subunits of the trimeric GTP-binding proteins, including Gs alpha. All of the biochemical characteristics of the purified ARF, including the lack of GTPase activity and the posttranslational myristoylation, are consistent with the derived sequences. Comparison of the ARF sequences to that of the chicken processed pseudogene (CPS-1), previously reported as a ras homologue, reveals that CPS-1 is actually an ARF-derived gene. These results demonstrate that ARF is a GTP-binding protein with structural features of both the ras and the trimeric GTP-binding protein families.

ADP-Ribosylation Factors↗
PubMed1986

The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein.

A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.

Adenosine Diphosphate Ribose↗
PubMed1986

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.

Animals↗
PubMed1985

Studies on the nature of the human platelet alloantigen, PlA1: localization to a 17,000-dalton polypeptide.

We have examined the biochemical properties of the human platelet alloantigen system, PlA, using a preparation enriched in plasma membrane glycoproteins (GPs) IIb and IIIa as the starting material. After confirming that GPIIIa contains the PlA epitope, attempts were made to distinguish the two allelic forms of PlA (A1 and A2) using electrophoretic techniques. Whereas no difference could be discerned in the mol. wt of GPIIIa extracted from A1/A1, A1/A2 or A2/A2 platelets by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis revealed a reproducible difference in the isoelectric point of GPIIIa derived from A2/A2 individuals. Treatment of GPIIIa with a combination of exo- and endoglycosidases resulted in apparently complete deglycosylation of the molecule, as indicated by its co-migration with chemically deglycosylated GPIIIa in SDS-PAGE. The enzymatically deglycosylated protein retained its full ability to react with anti-PlA1 antibodies. Tryptic digestion of GPIIIa resulted in the generation of a number of smaller polypeptides, including one of 17,000 daltons, that contained the PlA1 determinant. These results suggest that the carbohydrate moieties of GPIIIa are unimportant to the expression of the PlA system, and that the charge difference between the two allelic forms of GPIIIa may reflect a subtle amino acid difference(s) within a 17K polypeptide region of the GP.

Antigen-Antibody Reactions↗
PubMed1985

Reconstruction of platelet proteins into phospholipid vesicles. Functional proteoliposomes.

Platelet membrane glycoproteins IIb and IIIa were reconstituted into liposomes containing phosphatidylcholine. The reconstituted vesicles bound antiplatelet antibodies and showed specific binding to thrombin-activated platelets. Prostacyclin, a known inhibitor of thrombin-activated platelet aggregation, inhibited the binding of the proteoliposomes to thrombin-activated platelets. The reconstituted vesicles also specifically bound 125I-labeled fibrinogen. This binding was insensitive to ADP but dependent on calcium ions. These data indicate that platelet glycoproteins IIb and IIIa have been successfully reconstituted into phospholipid vesicles such that their behavior is similar to that in intact platelets.

Blood Platelets↗
PubMed1985

Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3.

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP-2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb-specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311-1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.

Animals↗
PubMed1985

Prevalence of hepatitis B antibodies in personnel at a children's hospital.

Antibody to hepatitis B core antigen and antibody to hepatitis B surface antigen were measured in serum samples from 825 personnel at St Louis Children's Hospital (702 were possibly at high risk of occupational hepatitis B virus (HBV) exposure and 123 were office personnel); 5.6% had positive findings for both antibodies, 5.6% had findings for antibody to hepatitis B surface antigens alone, and 1.3% had positive findings for antibody to hepatitis B core antigens alone. The group with positive findings for antibody to hepatitis B surface antigens alone did not have traditional risk factors for HBV infection, suggesting that this serologic finding may not be a reliable indicator of past HBV infection. After accounting for the effects of age, sex, and ethnicity, it was found that no occupational group had a significantly increased prevalence of HBV antibodies compared with prevalence in other personnel. In comparison with volunteer blood donors, only physicians older than 40 years of age had an increased HBV antibody prevalence. It is concluded that St Louis Children's Hospital has not been a high-risk environment for HBV exposure in recent years. However, caution is advised in generalizing these conclusions because other children's hospitals may serve a patient population at higher risk of HBV infection. Decisions regarding HBV immunization policy should take into consideration the fact that personnel at different hospitals may face markedly different risks of HBV exposure.

Adult↗
PubMed1984

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin.

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARF is an intrinsic membrane protein with Mr = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of Gs can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.

ADP-Ribosylation Factors↗
PubMed1984

ADP-ribosylation of Gs promotes the dissociation of its alpha and beta subunits.

We have utilized purified reactants and cofactors to examine the form of the stimulatory guanine nucleotide-binding regulatory component (Gs) of adenylate cyclase that serves as a substrate for ADP-ribosylation by cholera toxin; we have also investigated some of the consequences of that covalent modification. Activation of Gs with nonhydrolyzable analogs of GTP, which causes dissociation of its subunits, completely inhibits the toxin-catalyzed covalent modification. However, this effect cannot be explained by subunit dissociation, since activation of Gs by fluoride is not inhibitory and ADP ribosylation of the alpha (45,000-Da) subunit of Gs proceeds equally well in the presence and absence of the beta (35,000-Da) subunit. ADP-ribosylation of the alpha subunit of Gs decreases its apparent affinity for the beta subunit; however, the affinity of alpha and ADP-ribosyl-alpha for GTP appear to be approximately the same. ADP-ribosylation of Gs thus promotes the dissociation of its alpha and beta subunits. This effect may account for or contribute to the activation of adenylate cyclase by cholera toxin.

Adenosine Diphosphate Ribose↗
PubMed1984

ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation.

Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclic GMP-selective phosphodiesterase. Radiolabel from [adenylate-32P]NAD+ was incorporated specifically into the alpha subunit of purified transducin. Maximal levels of incorporation approximated 0.8 mol of ADP-ribose/mol of transducin. A peptide containing the ADP-ribosyl moiety was purified from a tryptic digest of radiolabeled transducin. This peptide was characterized by chemical and enzymatic procedures and by fast atom bombardment mass spectrometry. The primary structure of this peptide was Glu-Asn-Leu-Lys-Asn(ADP-ribose)-Gly-Leu-Phe. It is probable that the peptide originated from the carboxyl terminus of the alpha subunit and that the ADP-ribosyl moiety is attached by an N-glycosidic linkage to the asparagine residue. Transducin associated with retinal disc membranes is also ADP-ribosylated by cholera toxin. Cholera toxin and islet-activating protein sequentially catalyze the incorporation of 1.9 mol of ADP-ribose/mol of transducin, indicating two distinct sites of ADP-ribosylation within transducin.

Adenosine Diphosphate Ribose↗
PubMed1983

Purification of human platelet membrane glycoproteins IIb and IIIa using high-performance liquid chromatography gel filtration.

A method for purifying platelet membrane glycoproteins IIb and IIIa to homogeneity has been developed. The procedure involves high-pressure gel filtration chromatography using a TSK-4000SW column in the presence of sodium dodecyl sulfate. This technique is capable of rapidly preparing milligram quantities of each glycoprotein with greater than 90% recovery. The use of this technique should aid in defining the structural and functional properties of GPIIb and GPIIIa.

Blood Platelets↗
PubMed1983

Diseases transmitted by blood transfusion.

Except for post-transfusion hepatitis (PTH), the transmission of disease by blood transfusion appears to be very limited. There are only a few case reports documenting the transmission of malaria and other parasitic diseases, syphilis, or bacterial infections. The ability to eradicate PTH will depend on the development of sensitive-specific tests for the non-A, non-B agent(s). In the interim, two important steps that might be taken to reduce the incidence of PTH are the careful recruitment of blood donors and, most important, the judicious use of blood and its components.

Bacterial Infections↗