PROSTAGLANDINS
1977 WINTER PROSTAGLANDINCONFERENCE Vail, Colorado,March 27-29, 1977 SESSION I: BIOCBBMISTRY The 1977 Winter ProstaglandinConferenceopened with Bengt Samuelsson,Chairmanof the session,characterizingthe endoperoxidesas the "mothersubstances"in that they give rise to the thromboxanes,to PG12 and to the classical prostaglandins. The fir-etspeaker,Bill Lands of the Departmentof Biochemistryat the Universityof Michigan,reviewedcharacteristics of the cycle-oxygenase-reaction mechanism. In the action of the cycle-oxygenase, one fatty acid molecule,two oxygens,an enzyme and an activatorcome togetherto form a product. Attemptshave been made to subdividethe process. A peculiarityof cycle-oxygenase is its deactivationeven in the presenceof oxygen and arachidonicacid. Another unusual feature is the continualneed for an activatoras demonstrated by an initial lag phase and the additionof glutathione peroxidaaewhich stops the reactionof the cycle-oxygenase. Thus the activatoris a hydroperoxide,probably the endoperoxide. With inactivationof the glutathioneperoxidase, the reactionrecoveredenough to generateproducts to the extent of controls. When 5,8,11,14-eicosatetrynoic acid (TYA)was the substrate,no oxygen was consumed. Upon subsequentaddition of arachidonate,time was requiredbefore product formation was observed. These results showed the formationof a substratecontaining-activated complex prior to oxygenation. The molecularweight of purifiedcycle-oxygenase was 70,800-72,000daltons by SDS electrophoreais, but was 380,088by direct measurement;a tetrapeptidewas indicated. The cycle-oxygenasefrom seminalvesiclescontainsheme. When heme was removedby acidic organic extraction,enzyme activitywas lost. Heme was added to purifiedenzyme to study its significance. Maximum activitywas obtainedwith 0.5 hems per 70,008 dalton unit in additionto 0.5 units of non-hemeiron. An A2B2 tetra-peptidewith two subunits binding heme and two subunitscontainingnon-hemeiron was suggested. A non-hemsenzyme, soybean lipoxygenasehas some characteristics of the cycle-oxygenaaeand was therefore used to study the first reactionsof the cycle-oxygenase which could involve the non-heme subunits. The interaction of cycle-oxygenase with 11,14-eicosadienoic acid yields a C-11 hydroperoxide. This interactionwas consideredsimilar to that of the first steps in the interactionof the cyclooxygenasewith arachidonicacid. Unfortunately,heme was
JULY 1977 VOL. 14 NO. 1
181
PROSTAGLANDINS needed for the formation of the C-11 hydroperoxide. Consequently, soybean lipoxygenase cannot be used to study the first reactions of the cycle-oxygenase. Examination of the oxygen uptake did indicate a "pingpang" mechanism. The oxygen interacts with a different form of the enzyme from the substrate. The mechanism proposed has been outlined.
YEAok
EC2 +A O2 1t
t
E 2A\m....“;*
4 x to-2
6 x lo’2
9 x 10’2
op
4 x 10-3
3 x 10-2
3 x 10-2
3 x 10’3
1 x 10’2
2 x 10’2
2 x 10-3
9 x 1o-2
0 x 1o-2
0
\ . %N
0 *
Y
G$
O>...F
220
RELATIVE
, ,
JULY 1977 VOL. 14 NO. 1
PROSTAGLANDINS
1. NBS,
13,14-dehydro-PGFp
CH CN 2.
CH2N2
I
i3,14-dehydro-IT’
13,14-dehydro-I’
,
(5490) DBN
DEN
I
Zhr,
Zhr,
1OOW
-P +-
lOO=‘C
Da%
0
z
i
Q ~_y” 2
HOI
zti
0, 2
no‘
%I 13,14-dehydro-X
13,14-dehydro-PG12
Fried agrees with Johnson (rather than Corey) as to the configuration at C-5 and C-6 of the major cyclization product based on the same mechanistic rationale. An additional support for the exo -assignment for the major product comes from studies of the dehydrohalogenation. The formation of the Z isomer from the major exo product can be rationalized as internal proton abstraction. -
--a
z-PGl* methyl
ester
In the endoseries, such a mechanism is impossible and the more accessim C-4 hydrogen is abstracted directly. As a test of this rationale the dehydrohalogenation of the THP derivative was examined. In this case only the A 4,5 product is obtained from the major exo product. Similar dehydrohalogenation of the enloproduct, incontrast to the DBU conditions on the hydroxy compo%, afforcgthe 11,15-bis-THP derivative of PGI2 methyl ester. The exo product (as the acid) affords in addition the olefinic elimination product, a lactone (XIII) on base treatment.
JULY 1977 VOL. 14 NO. 1
221
PROSTAGLANDINS
At pH 7.5 the methyl ester of 13,14 dehydro-PG12 was stable for a couple of days at 250C. The free acid of PGI2 is highly unstable and considering the ease of lactone formation, discussed previously, the acid group could internally catalyze the decomposition to C-keto-PGFla. The biological activity of the 13,14-dehydro-PGI2 methyl ester was investigated by Samuelsson and colleagues and presented in the abstracts of the Wall Poster Presentation. At 2-10 nmoles, platelet aggregation and serotonin release were inhibited. Fei en of Kadowitz's group described biological data obtained with T5;9h-dehvdro-PGF?a methvl ester and the 1.5-lactone svntheSized by Fried-et al. &Measuring renal blood flow of a perfused canine kidney, E D2 and A2 were vasodilators (E2 0.03 to 0.3 lJg, while D2 and A2 2Ad one-fifth the activity) and PGH2 (100 to 600 ng) and arachidonate (0.1 to 1 ms) had a biphasic effect of a momentary constriction followed by vasodilation which was variable from animal to animal. The 13,14-dehydro-PG12 was approximately as potent as PGE2 which is cansistent with results obtained by Fletcher in Rhesus monkeys. The 1,5-lactone (30 ug) did produce some vasodilation which was greater than the response to PGF2a. The 13,14-aehydro-analogue of PGF2a methyl ester was about as active as PGE2 and the lactone had some activitv on mesenteric blood flow. Next, S. Hammarstrom of-the Karolinska group presented data of the activity of 13,14-dehydro-PG12 methyl ester in increasing CAMP in 3T3 fibroblasts.* For,more information refer to the wall posters in the June Issue of PRGSTAGLANDINS.
Reported by Elizabeth M.K. Leovey
222
JULY 1977 VOL. 14 NO. I
1977 WINTER PROSTAGLANDINCONFERENCE Vail, Colorado,March 27-29, 1977 SESSION I: BIOCBBMISTRY The 1977 Winter ProstaglandinConferenceopened with Bengt Samuelsson,Chairmanof the session,characterizingthe endoperoxidesas the "mothersubstances"in that they give rise to the thromboxanes,to PG12 and to the classical prostaglandins. The fir-etspeaker,Bill Lands of the Departmentof Biochemistryat the Universityof Michigan,reviewedcharacteristics of the cycle-oxygenase-reaction mechanism. In the action of the cycle-oxygenase, one fatty acid molecule,two oxygens,an enzyme and an activatorcome togetherto form a product. Attemptshave been made to subdividethe process. A peculiarityof cycle-oxygenase is its deactivationeven in the presenceof oxygen and arachidonicacid. Another unusual feature is the continualneed for an activatoras demonstrated by an initial lag phase and the additionof glutathione peroxidaaewhich stops the reactionof the cycle-oxygenase. Thus the activatoris a hydroperoxide,probably the endoperoxide. With inactivationof the glutathioneperoxidase, the reactionrecoveredenough to generateproducts to the extent of controls. When 5,8,11,14-eicosatetrynoic acid (TYA)was the substrate,no oxygen was consumed. Upon subsequentaddition of arachidonate,time was requiredbefore product formation was observed. These results showed the formationof a substratecontaining-activated complex prior to oxygenation. The molecularweight of purifiedcycle-oxygenase was 70,800-72,000daltons by SDS electrophoreais, but was 380,088by direct measurement;a tetrapeptidewas indicated. The cycle-oxygenasefrom seminalvesiclescontainsheme. When heme was removedby acidic organic extraction,enzyme activitywas lost. Heme was added to purifiedenzyme to study its significance. Maximum activitywas obtainedwith 0.5 hems per 70,008 dalton unit in additionto 0.5 units of non-hemeiron. An A2B2 tetra-peptidewith two subunits binding heme and two subunitscontainingnon-hemeiron was suggested. A non-hemsenzyme, soybean lipoxygenasehas some characteristics of the cycle-oxygenaaeand was therefore used to study the first reactionsof the cycle-oxygenase which could involve the non-heme subunits. The interaction of cycle-oxygenase with 11,14-eicosadienoic acid yields a C-11 hydroperoxide. This interactionwas consideredsimilar to that of the first steps in the interactionof the cyclooxygenasewith arachidonicacid. Unfortunately,heme was
JULY 1977 VOL. 14 NO. 1
181
PROSTAGLANDINS needed for the formation of the C-11 hydroperoxide. Consequently, soybean lipoxygenase cannot be used to study the first reactions of the cycle-oxygenase. Examination of the oxygen uptake did indicate a "pingpang" mechanism. The oxygen interacts with a different form of the enzyme from the substrate. The mechanism proposed has been outlined.
YEAok
EC2 +A O2 1t
t
E 2A\m....“;*
4 x to-2
6 x lo’2
9 x 10’2
op
4 x 10-3
3 x 10-2
3 x 10-2
3 x 10’3
1 x 10’2
2 x 10’2
2 x 10-3
9 x 1o-2
0 x 1o-2
0
\ . %N
0 *
Y
G$
O>...F
220
RELATIVE
, ,
JULY 1977 VOL. 14 NO. 1
PROSTAGLANDINS
1. NBS,
13,14-dehydro-PGFp
CH CN 2.
CH2N2
I
i3,14-dehydro-IT’
13,14-dehydro-I’
,
(5490) DBN
DEN
I
Zhr,
Zhr,
1OOW
-P +-
lOO=‘C
Da%
0
z
i
Q ~_y” 2
HOI
zti
0, 2
no‘
%I 13,14-dehydro-X
13,14-dehydro-PG12
Fried agrees with Johnson (rather than Corey) as to the configuration at C-5 and C-6 of the major cyclization product based on the same mechanistic rationale. An additional support for the exo -assignment for the major product comes from studies of the dehydrohalogenation. The formation of the Z isomer from the major exo product can be rationalized as internal proton abstraction. -
--a
z-PGl* methyl
ester
In the endoseries, such a mechanism is impossible and the more accessim C-4 hydrogen is abstracted directly. As a test of this rationale the dehydrohalogenation of the THP derivative was examined. In this case only the A 4,5 product is obtained from the major exo product. Similar dehydrohalogenation of the enloproduct, incontrast to the DBU conditions on the hydroxy compo%, afforcgthe 11,15-bis-THP derivative of PGI2 methyl ester. The exo product (as the acid) affords in addition the olefinic elimination product, a lactone (XIII) on base treatment.
JULY 1977 VOL. 14 NO. 1
221
PROSTAGLANDINS
At pH 7.5 the methyl ester of 13,14 dehydro-PG12 was stable for a couple of days at 250C. The free acid of PGI2 is highly unstable and considering the ease of lactone formation, discussed previously, the acid group could internally catalyze the decomposition to C-keto-PGFla. The biological activity of the 13,14-dehydro-PGI2 methyl ester was investigated by Samuelsson and colleagues and presented in the abstracts of the Wall Poster Presentation. At 2-10 nmoles, platelet aggregation and serotonin release were inhibited. Fei en of Kadowitz's group described biological data obtained with T5;9h-dehvdro-PGF?a methvl ester and the 1.5-lactone svntheSized by Fried-et al. &Measuring renal blood flow of a perfused canine kidney, E D2 and A2 were vasodilators (E2 0.03 to 0.3 lJg, while D2 and A2 2Ad one-fifth the activity) and PGH2 (100 to 600 ng) and arachidonate (0.1 to 1 ms) had a biphasic effect of a momentary constriction followed by vasodilation which was variable from animal to animal. The 13,14-dehydro-PG12 was approximately as potent as PGE2 which is cansistent with results obtained by Fletcher in Rhesus monkeys. The 1,5-lactone (30 ug) did produce some vasodilation which was greater than the response to PGF2a. The 13,14-aehydro-analogue of PGF2a methyl ester was about as active as PGE2 and the lactone had some activitv on mesenteric blood flow. Next, S. Hammarstrom of-the Karolinska group presented data of the activity of 13,14-dehydro-PG12 methyl ester in increasing CAMP in 3T3 fibroblasts.* For,more information refer to the wall posters in the June Issue of PRGSTAGLANDINS.
Reported by Elizabeth M.K. Leovey
222
JULY 1977 VOL. 14 NO. I
