Genetica

RELATION BETWEEN INCORPORATION

PHOSPHATE OF LABELED PROTEINS

ENERGY DONORS AND AMINO ACIDS INTO *
B. KELLER Memorial Hospi’tal Hospital, Boston,

BY PAUL (From

C. ZAMECNIK

AND ELIZABETH

the Medical Laboratories of the Collis P. Huntington Harvard University, at the Massachusetts General Massachusetts) (Received for publication, January

of

27, 1954)

Incorporation oflabeled amino acids into proteins in cell-free systems has been reported from several laboratories (l-5) in recent years. The small extent of incorporation has, however, made it difficult to explore the relationship of this process to energy-yielding mechanisms and to protein synthesis in viva. In this context, the term “incorporation” has come to imply a, tight bonding of labeled amino acid toprotein, with reservation of judgment as to whether or not this represents the cu-peptide bonding of protein synthesis. As a result of the finding of Bucher (6) that gentle homogenization of rat liver preserves in the cell-free fraction the ability to incorporate C14acetate into cholesterol, we were encouraged to try a similar system for our purpose (7), and the present report is an outgrowth of thisstudy.
EXPERIMENTAL

Downloaded from www.jbc.org by guest, on February 22, 2013

MateriuZs-nL-Leucine-1-C14, m-alanine-l-C14, glycine-1-C14, m-valine-lC14, and nL-a-aminobutyric acid-l-C?4 were synthesized by Dr. Robert B. Loftfield (8) and were kindly furnished at specific activities ranging from 0.8 to 10 mc. per mM. DPN,’ ATP, ADP, and AMP were recent Pabst or Sigma preparations. HDP and3-PGA were obtained from Schwarz, both of them as barium salts. Phosphocreatine, sodium salt, was obtained from Sigma, and assayed as 95 per cent phosphocreatine tetrahydrate, by measuring the difference in phosphate in the Fiske-Subbarow (9) method and a modified Lowry-Lopez (10) method. Phosphorylenolpyruvic acid, silver-barium salt, was kindly furnished by Dr. Henry Sable. For a gift ofDL-ethionine
* Supported by grants-in-aid from the American Cancer Society and the Atomic Energy Commission. This is publication No. 820 of the Cancer Commission of Harvard University. * The following abbreviations have been used : ATP, adenosinetriphosphate; ADP, adenosinediphosphate; AMP, adenosine-5’-phosphate; HDP, hexose diphosphate; 3-PGA, 3-phosphoglyceric acid; PPA, phosphorylenolpyruvic acid; PC,phosphocreatine; CSH, cysteine; DPN, diphosphopyridine nucleotide; CoA, coenzyme A. 337

338

ATP

AND

AMINO

ACID

INCORPORATION

we are indebted to Dr. V. du Vigneaud. Neurospora DPNase was a gift of Dr. Nathan 0. Kaplan. Crystalline ribonuclease was obtained from Worthington. The CoA was generously provided by Dr. F. Lipmann. Preparation of Liver Extract-2 month-old Wistar ratswere used. 2 to 4 gm. of liver were minced for 15 to 20 seconds with scissors, in 1 ml. of the medium to be used, in a glass vessel surrounded by ice. The mince was transferred to a glass homogenizing tube, kept in an ice bath at O”, and homogenized (11) for 15 to 25 seconds with an ice-cold glass pestle, in a total of 2.5 times its weight of medium. Much deviation up or down from this proportionproduced less favorable results. The difference in diameter of pestle and tube was usually 0.6 mm. and the rate of revolution of the pestle 600 r.p.m. The resulting partial homogenate was centrifuged immediately at 0” for 5 minutes at approximately 500 X g (when a mitochondrium-rich system was used) or at 5000 X g. The upper portion of the supernatant fluid was carefully pipetted off and usedimmediately for the incorporation experiments. It was found important to centrifuge the homogenate immediately and to keep it at 0” throughout the steps prior to incubation. In some experiments the 5000 X g supernatant fluid was separated into a microsome-rich fraction and a fraction containing the soluble cell proteins by centrifugation for 30 minutes at 0’ and 105,000 X g (R average) in a Spinco...