Identification of spinach farnesyl protein transferase. Dithiothreitol as an acceptor in vitro.
Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.
Parmryd
I
I
Arrhenius Laboratories for Natural Sciences, Biochemistry Department, Stockholm University, Sweden.
Shipton
C A
CA
Swiezewska
E
E
Andersson
B
B
Dallner
G
G
D019883:Alkyl and Aryl Transferases; D015153:Blotting, Western; D002712:Chlorides / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism* / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005204:Farnesol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D013058:Mass Spectrometry; D008798:Mevalonic Acid / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011126:Polypropylenes / Q000378:metabolism; D017368:Protein Prenylation; D012717:Sesquiterpenes; D018724:Spinacia oleracea / Q000201:enzymology*; D013696:Temperature; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D011126:Polypropylenes; D012717:Sesquiterpenes; D017967:Zinc Compounds; D015636:Magnesium Chloride; D005204:Farnesol; C004808:farnesyl pyrophosphate; C016837:zinc chloride; D004492:Edetic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D008798:Mevalonic Acid; D004229:Dithiothreitol