Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells

Barbara C. Kunz, Kimberly A. Muczynski, Catherine Welsh, Sally J. Stanley, Su Chen Tsai, Ronald Adamik, Patrick P. Chang, Joel Moss, Martha Vaughan

Research output: Contribution to journalArticle

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Abstract

ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.

Original languageEnglish
Pages (from-to)6643-6648
Number of pages6
JournalBiochemistry
Volume32
Issue number26
StatePublished - Dec 1 1993
Externally publishedYes

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ADP-Ribosylation Factors
Sf9 Cells
Insects
Cholera Toxin
Guanosine Triphosphate
Adenosine Diphosphate
Escherichia coli
Baculoviridae
Proteins
Guanine Nucleotides
Ion Exchange Chromatography
Myristic Acid
Isoelectric Focusing
Protein Transport
Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kunz, B. C., Muczynski, K. A., Welsh, C., Stanley, S. J., Tsai, S. C., Adamik, R., ... Vaughan, M. (1993). Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells. Biochemistry, 32(26), 6643-6648.

Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells. / Kunz, Barbara C.; Muczynski, Kimberly A.; Welsh, Catherine; Stanley, Sally J.; Tsai, Su Chen; Adamik, Ronald; Chang, Patrick P.; Moss, Joel; Vaughan, Martha.

In: Biochemistry, Vol. 32, No. 26, 01.12.1993, p. 6643-6648.

Research output: Contribution to journalArticle

Kunz, BC, Muczynski, KA, Welsh, C, Stanley, SJ, Tsai, SC, Adamik, R, Chang, PP, Moss, J & Vaughan, M 1993, 'Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells', Biochemistry, vol. 32, no. 26, pp. 6643-6648.
Kunz BC, Muczynski KA, Welsh C, Stanley SJ, Tsai SC, Adamik R et al. Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells. Biochemistry. 1993 Dec 1;32(26):6643-6648.
Kunz, Barbara C. ; Muczynski, Kimberly A. ; Welsh, Catherine ; Stanley, Sally J. ; Tsai, Su Chen ; Adamik, Ronald ; Chang, Patrick P. ; Moss, Joel ; Vaughan, Martha. / Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells. In: Biochemistry. 1993 ; Vol. 32, No. 26. pp. 6643-6648.
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