The action of prostaglandin 15-hydroxydehydrogenase on various prostaglandins

581

SHORT COMMUNICATIONS BBA 53241 The action of prostaglandin 15-hydroxydehydrogenase prostaglandins

on various

The specificity of the prostaglandin I5-hydroxydehydrogenase from swine lung has been studied by ANGGARD AND SAMUELSSON~on most of the naturally occurring prostaglandins and also on a number of compounds with widely divergent structures. We have extended these studies to a variety of prostaglandins not found in nature, namely the biologically active prostaglandins derived from the newly discovered essential fatty acids with an odd number of carbon atoms2 and some prostaglandins derived from shorter- and longer-chain homologues of dihomo-y-linolenic acid (20 : 3) and arachidonic acid (20: 4)3. All prostaglandins E used were prepared biosynthetically from the corresponding fatty acids (cj. Table I). Prostaglandin A, and prostaglandin A, were prepared from prostaglandin E, and prostaglandin E, by heating at 60” for 16 h in 90% acetic acid4. An enzyme preparation, derived from swine lung by essentially the same method as described by ANGGARD AND SAMUELSSON~,was used. Chromatography on hydroxylapatiteand DEAE-Sephadex was not applied as it gave a considerable loss in total activity and only a small gain in specific activity. The result of our last purification step, Sephadex G-100 gel filtration, is given in Fig. I. Fractions 58-64 were combined and used as enzyme preparation. The protein content of this fraction was determined by the biuret method6. Incubations were carried out on such a scale that the I5-ketoprostaglandins produced could be determined by spectrophotometrye. The assays were performed in the following way: 1.43 /[moles of prostaglandin (i.e. for prostaglandin E,, 500 pg) and 6.5 mg protein were incubated with 5 /‘moles NAD in 7.5 ml phosphate buffer (0.1 M, pH 7.3) at 44’. After IO, 20, 40 and 60 min, samples

Fig. I. Sephadex G-100 column eluted with 0.1 M phosphate buffer. O---O, protein (Azsonm o--o, amount of r5-ketoprostaglandin E, formed (A,,, & by incubation of 0.5 mg protein. Biochim.

Biofihys.

Acta,

187 (1969)

581-583

);

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SHORT COMMUNICATIONS

of I ml were taken and acidified to pH 3 with a citric acid solution, while the 15ketoprostaglandin E was extracted with 3 vol. of diethyl ether. The combined ethereal extracts were washed with water until neutral and evaporated to dryness. The residue was dissolved in I ml methanol and the amount of r5-ketoprostaglandin E determined by diluting an aliquot to 2.5 ml with methanol in a spectrophotometric cell, adding 0.5 ml of 0.6 M KOH in methanol-water (3 : I, v/v) and measuring the increase of absorbance at 500 nm. A maximum was reached after 5-7 min, depending on temperature, after which the absorbance again decreased slowly. From the maximal absorbance

the amount

of r5-ketoprostaglandin

E

can

be calculated

(AA,,,,,

is

about 3.8 for I pmole r5-ketoprostaglandin E, in 3 ml final volumee). In a blank incubation with boiled enzyme, no r5-ketoprostaglandin E could be detected. For several samples the spectrum was determined when the maximum value for the absorbance

at 500 nm had been reached

tion peak at 500 nm was present.

20

LO

to see whether

a substance

This indeed appeared

to be true.

with an absorp-

60

Fig. 2. Formation of some r5-ketoprostaglandins prostaglandin I5-hydroxydehydrogenase. A-A, din E,; o--o o-homoprostaglandin E,.

Two series of incubations

E during different periods

w-norprostaglandin

were performed

with separately

of incubation with E,; o---n, prostaglan-

prepared

batches

of

enzyme. Some of the prostaglandins were tested with both enzyme preparations so that the results give an impression of the reproducibility of the method. For each prostaglandin tested, the amount of r5-ketoprostaglandin E produced was plotted against the time of incubation (some examples are given in Fig. 2). Straight lines were obtained except for prostaglandin A,. In this case it is rather difficult to determine the amount of r5-ketoprostaglandin A accurately, as the maximum absorption is obtained immediately upon the addition of KOH and starts to decrease at once. The rates of conversion of the various prostaglandins were compared with those of prostaglandin E, which were taken as IOO (see Table I). As the amounts of r5-ketoprostaglandins E formed are proportional to the incubation times, the reaction rates are constant for periods up to I h. Hence the reaction rates for the different prostaglandins can be compared by determining the amounts of r5-ketoprostaglandins E formed. From Table I it can be seen that the prostaglandins tested are oxidized by the r5-hydroxydehydrogenase at rates which are of the same order of magnitude. It can be concluded that a fixed length of the carboxyl and/or the alkyl chain of the prostaBiochinz. Biophys.

Acta,

187 (1969)

581-583

583

SHORT COMMUNICATIONS TABLE

I

RELATIVE GLANDINS

ACTIVITIES

OF PROSTAGLANDIN

15-HYDROXYDEHYDROGENASE

ON VARIOUS PROSTA-

All double bonds in the precursor acids from which the prostaglandins are derived by biosynthesis, have cis-configuration. The short notation indicates position of double bonds-number of carbon atoms: number of double bonds; the o-numbering system refers to the position of the first double bond calculated from the methyl end of the acid. Rate of conversion relative to prostaglandin E,

Fatty acid precursor

Prostaglalzdin incubated

Expt. 2

Expt. I a-Norprostaglandin E, Prostaglandin E, cr-Homoprostaglandin E, w-Norprostaglandin E, o-Homoprostaglandin E, cc,,%Dinorprostaglandin E, or-Norprostaglandin E, Prostaglandin E, a-Homoprostaglandin E, a-Dihomoprostaglandin E, o-Norprostaglandin E, o-Homoprostaglandin E, Prostaglandin E, Prostaglandin A, Prostaglandin A,

glandin

is not

biologically

required

active

“unnatural”

but

The

II, I4-I9:3 (w5) II, 14-21:3 (W7) 6, g, IZ-18:4 (06) 7, IO, I3-I9:4 (06) 8, II, 14-20:4 (06) 6, g, 12, 15-21:4 (06) 7. IO, 13, 16-22:4 (06) 5, 8, II, 5. 8, II. 5, 8, II,

for

(w5) (W7)

conversion.

active

The

The

technical

100 -

46 ‘07 35 46 67 78 75 96 85 -

87 80 34 -

52 50

33

are perhaps of

prostaglandins

as the ordinary assistance

rates

results

-5,8,rI,rq-tetraenoic

as rapidly

skillful

I4-19:4 I4-2I:4

‘4, 17-20:5 (W‘3) 8, II, I4-20:3 (06) 5. 8, II, 14-20:4 (~6)

biologically and

33 100 72 100 66 -

8, 8, 3, 4, 5,

prostaglandins.

cis-8,Ir,rq-trienoic be inactivated

7. 10, I3-lg:3 (~6) 8, II, 14-20:3 (~6) g, 12, 15-21: 3 (~6)

this

(derived

acids)

slightly

higher

investigation from

administered

for

indicate

the that

odd-numbered

all-

to an

will

animal,

prostaglandins.

of Mr.

W.

C. VAN

EVERT

is gratefully

acknow-

ledged.

U&lever Research Laboratory, Vlaardingen

(The Netherlands)

H. VONKEMAN D.

H. NUGTEREN

D. A. VAN DORP I E. ANGGARD AND B. SAMUELSSON, Arkiv Kemi, 25 (1966) 293. 2 R. K. BEERTHUIS, D. H. NUGTEREN, H. J. J. PABON AND D. A. VAN DORP, Rec. Trav. Chim.,

87 (1968) 461. 3 C. B. STRUIJK, R. K. BEERTHUIS, H. J. J. PABON AND D. A. VAN DORP, Rec. Trav. Chim., 85 (1966) 1233. 4 E. G. DANIELS, J. W. HINMAN, B. A. JOHNSON, F. P. KUPIECKI, J. W. NELSON AND J. E. PIKE, B&hem. Biophys. Res. Commun., 21 (fg65) 413. 5 S. P. COLOWICKAND N. 0. KAPLAN, Methods in Enzymology, Vol. 3. Academic Press, New York, 1957, P. 450. 6 D. H. NUGTEREN, R. K. BEERTHUIS AND D. A. VAN DORP, Rec. Trav. Chim., 85 (1966) 405. Received

July

7th,

1969

Biochim. Biophys. Acta, 187 (1969) 581-583