A Spectrophotometric Assay for Thiogalactoside Transacetylase

THE JOURNAL OF BIOLOGICAL Vol. 240, No. 1, January Printed in U S.A.

CHEMISTRY 1965

A Spectrophotometric DAVID

Assay

H. ALPERS,*

for Thiogalactoside

STANLEY

H. APPEL,~.

AND GORDON

Transacetylase

M. TOMKINS

From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Bethesda, Maryland 20014 (Received Thiogalactoside Acetyl-CoA

transacetylase,

+ isopropyl

isopropyl

+ p-n-thiogalactoside

(1)

EXPERIMENTAL

acid is followed

3, 1964)

spectro-

PROCEDURE

Materials-Bacterial strains used were E. coli K12, strains 200P Flat (i+z+y-/F i+z+y+), AB 1105 (i+z+y+), 2ZOlc (i-z+y-), and 2EOlc (i-z+y-); E. coli B (i+z+y+) ; Xhigella dysenteriae Sh 60 (i+z+ydel); Salmonella typhimurium LT2 (lac-); and Serratia marcescens.’ DTN2 was purchased from the Aldrich * Present address, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. t Present address, Department of Neurology, Duke University Medical School, Durham, North Carolina. 1 We would like to thank the following persons for these bacterial strains: Drs. F. Jacob, A. J. Clark, T. Fukasawa, W. Dreyer, C. B. Anfinsen, and A. Weissbach.

2 The abbreviations used are : DTN, 5,5’-dithiobis (2-nitrobenzoic acid); IPTG, isopropyl P-n-thiogalactoside; acetyl-IPTG, acetylated isopropyl fi-o-thiogalactoside; i, the gene controlling inducibility in the lactose operon; z, the structural gene for p-galactosidase; y, the structural gene for galact,oside permease; NPG, o-nitrophenyl fl-o-galactoside. 10

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was first detected by Zabin, Kepes, and Monod in extracts of Escherichia co& (I), and has recently been crystallized (2). The enzyme is considered to be a product of the lactose operon. During induction of P-galactosidase, transacetylase activity increases proportionately. Furthermore, in certain galactoside permeaseless mutants (y-), low levels of transacetylase are found. Its physiological function and importance are unknown, alt’hough it has been suggested that it may be related to galactoside transport (1). Investigations of transacetylase have been limited by the relatively cumbersome assays presently in use, which are based either on the formation of an acetylhydroxamate from the acetylated thiogalactoside (3) or on the detection of radioactive acetylated thiogalactosides (4). The present communication presents a rapid spectrophotometric assay which is about 100 times more sensitive than the hydroxamate assay, and which can be used with crude bacterial extracts. This new method is based on a disulfide interchange reaction between the CoA liberated from acetyl-CoA and the dithiobis(2nitrobenzoic acid) reagent described by Ellman (5) (Reaction 2).

The appearance of thionitrobenzoic photometrically at 412 mM.

July

Chemical Company, Milwaukee. Acetyl-CoA, IPTG, thiomethyl @-n-galactoside, and melibiose were obtained from Mann Research Laboratories, New York. Methyl phenyl thiogalactosides and methyl phenyl glucosides were gifts from Dr. N. Richtmeyer; acylpantetheine derivatives from Dr. R. Vagelos; acetylglutathione from Dr. W. W. Kielley; hexosamines from Dr. V. Ginsburg; and phosphorylated hexoses from Dr. G. Ashwell. NPG and acetyl phosphate were purchased from Sigma Chemical Company, St. Louis, and cat,alase was obtained from Worthington Biochemical Corporation, Freehold, New Jersey. Growth of Bacteria and Preparation of Extracts-Bacteria were grown at 37” in a New Brunswick rotary shaker in synthetic Medium 3XD (6) containing 0.9% glycerol and 0.5 pg of thiamine hydrochloride per ml. Most of the experiments to be described below were performed with extracts of E. coli 200P Flat. The doubling time of this organism was 50 minutes under the above conditions. Extracts were prepared from log phase cells grown with 0.5 mM IPTG for 1 to 4 hours until they reached a density of 1 to 3 x log cells per ml. The bacteria were then centrifuged at 12,000 x g for 15 minutes and resuspended in They 0.05 M Tris, pH 7.8, at one-tenth the original volume. were then chilled and disrupted at 4” with a Sonifier (Branson Instrument Company, Stamford, Connecticut) at a setting of 3 amperes for 30 seconds. The sonic extract was centrifuged at 30,000 x g for 20 minutes, and the pellet was discarded. Extracts from other bacteria were similarly prepared. Assays-p-Galactosidase was assayed at 25” by a modification of the method of Pardee, Jacob, and Monod (7) with 0.003 M NPG in a mixture of 0.1 hf sodium phosphate, pH 7.0, 0.001 M MgC12, and 0.01 M /%mercaptoethanol. The reaction was terminated by the addition of 0.5 volume of 1 M sodium carbonate, and the color was read at 420 rnp in cells of IO-mm light path in a Beckman DU spectrophotometer. The transacetylase assay described below was carried out in cells of IO-mm light path in a Gilford recording spectrophotometer, model 2000, at 412 mp. Sodium EDTA, pH 7.0, 0.1 mM, was routinely included in reaction mixtures. Acetyl-CoA and acetyl phosphate were estimated by the hydroxamate assay of Lipmann and Tuttle (8).

which catalyzes the reaction

P-n-thiogalactoside

CoA + acetylated

for publication,

January

D. H. Alpers, S. H. Appel, and G. M. Tomkins

1965 RESULTS

AND

DISCUSSION

.400

I

I

.800 t

7

‘2

.350

,300 ’

’

’

’

’

’

’

I .05

I .06

I .07

I .08

P.250 5 1.200 z.

.

150 .

.I00 .050