Richard A. Kahn

The ras superfamily of GTP-binding proteins: guidelines on nomenclature.

therasoncogeneproteinshasbeendescribed(1-4).Thenumberofmembersofthissuperfamily identified hasincreaseddra-matically inrecentyears,andthenumber ofdescriptivetermsornamesusedtodenotetheseproteinshasincreasedalmostasdramatically aswell.During therecentFASEBSummer ResearchConference on“LowMolecular WeightGTPBinding Proteins:’aninformal sessiononnomencla-turewasheldinanefforttoestablishguidelinesforthelitera-tureinthisrapidlyexpanding field,aswellasinthenamingofnewgenesandproteins. Theneedforsuchasessionclearlyresultsfromtheabundance ofsequenceinformationonnewgenesandproteinsandtherelativepaucityofinfor-mationregarding proteinfunction, acommonthemeinthebiological sciencestoday.Thediscussion focusedonthreemainquestions:

Detection of the major pertussis toxin substrate of human leukocytes with antisera raised against synthetic peptides

Antisera raised against the carboxy‐terminal decapeptide (KENLKDCGLF) of transducin‐α detected the 40 kDa, major pertussis toxin substrate of human neutrophils. The antisera also detected this protein in undifferentiated HL‐60 and U937 cells, and revealed an approx. 2‐fold increase in protein/mg membrane protein with differentiation into mature phagocytic cells. The results provide direct immunochemical evidence for the presence of a novel, pertussis toxin‐sensitive guanine nucleotide‐binding protein in human leukocytes.

Bacterial expression, purification, andin vitro N-myristoylation of HIV-1 pl7gag

The coding region of the N-terminal 17-kDa portion of HIV-1 Pr55gag (p17gag) was cloned into the pET-3c expression vector and was used to overexpress HIV-1 p17gag in Escherichia coli. Induction of the transformed bacteria caused the accumulation of a 17-kDa polypeptide in the soluble cell fraction which was released by sonication in hypotonic nondetergent buffer. The 17-kDa polypeptide was purified by ammonium sulfate precipitation and successive chromatography on G-75 Sephadex, DEAE-Sephacel, and S-Sephadex. The final product was purified 12-fold with about a 16% recovery from the original soluble cell lysate and was judged to be 97+% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting with two different antibodies confirmed the identify of the purified 17-kDa polypeptide as authentic p17gag. In the presence of myristoyl-CoA and bovine brain N-myristoyl-transferase, p17gag was quantitatively N-myristoylated in vitro with a pseudo-first-order rate constant of 4.7 +/- 1.0 x 10(-3) min-1, but with only about 3% of the catalytic efficiency of N-myristoylation of a 16-residue peptide homologous to the N-terminus of p17gag. The myristate group in the N-myristoylated p17gag was stable to treatment with detergent and hydroxylamine consistent with a covalent N-acyl-amide linkage. The N-myristoylglycyl linkage was confirmed by partial acid hydrolysis and identification of the p-nitrobenzylazlactone derivative of the resulting N-myristoylglycine by high-performance liquid chromatography.

PURIFICATION OF A SYNTHETIC MYRISTYLATED PEPTIDE BY COUNTER-CURRENT CHROMATOGRAPHY

A preparative purification of myristyl-Gly-Asn-Ile-Phe-Ala-Asn-Leu-Phe-Lys-Gly-Leu-Phe-Gly-Lys-Lys-Glu -NH2 was accomplished using the multi-coil counter-current chromatograph. A partition coefficient was determined in the n-butanol-acetic acid-water (4:1:5) system. Chromatographic runs were made in this system and one modified with ethyl acetate. The peptide material showed anomalous elution behavior due to its surfactant properties. It was found that by loading the sample exclusively in the stationary phase, satisfactory retention of the compound occurred. Finally, conditions utilizing the upper phase as the mobile phase successfully separated the impurities.