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On the functional significance of multiple agonist-binding sites in receptors
9th Meeting
c21
81
TRANSFER OF BETWEEN MICROSOMBS IN RAT BRAIN
of the ESN
82 PHOSPHATIDYLSERINE AND MITOCHONDRIA
0. Arienti, E. Carlini and L. Corazzi Istituto di Biochimica e Chimica Medica, de1 Giochetto, 06100 PERUGIA (Italy)
ON TEE FUNCTIONAL SIGNIFICANCE MULTIPLE AGONIST-BINDING SITES RECEPTORS. Via
Phosphatidylscrine in (PS) is synthesized brain microsomes through base-exchange. Yet, its decarboxylation to phosphatidylethanolamine (PE) occurs on the inner mitochonof the transdrial membrane. The mechanism fer from endoplasmic reticulum to mitochondria is still a debated question. Indeed, it has been ascribed to energy-dependent systems, to collision, or to other mechanisms. To characterize the transport of PS between rat brain subcellular fractions, we reconstituted in vitro a system able to synthesize and dacarboxylate PS. Brain homogenates subcellular and fractions were prepared using standard procedures and incubated in the presence of Caa+ and radioactive serine (base-exchange conditions) or in a medium free of Cd+ (PS decarboxylase conditions).
Rat brain homogenates could synthesize and decarboxylate PS; the same was true for a mixture composed of purified mitochondria and microsomes, that no soluble indicating factors were necessary for the transfer and bearing out a collision-based transport. The PS newly synthesized by base exchange was preferentially transferred, infeered examininp the dioactivities of micrcsomal
as can be specific raand mitochon-
drisl lipids. 83 PLATELET-ACTIVATING FACTOR (PAF) SYNTHESIZING RAT ENZYMES IN CENTRAL SYSTEM: NERVOUS PROPERTIES AND DISTRIBUTION. E. Francescangeli, A. Boila and G. Goracci. Institute di Biochimica e Chimica Medica, Universita di Perugia, Perugia, Italy. PAF is a lipid mediator which is present in the nervous tissue and is involved in physiological functions and in pathological situations (Goracci, 1990). TWO metabolic routes have been described for the synthesis of PAF in a variety of tissues and cell the de types: IlO”0 and the pathway remodeling pathway. Previously, we have demonstrated that rat br;ain may synthesize PAF by both pathways (Francescangeli and 1989; Goracci and Francescangeli, Goracci, 1991). PAF-synthesizing phosphocholinetransferase (PAF-PCT) catalyzes the last reaction of the de nova synthesis whereas lyso-PAF acetyltransferase (LysoPAF-AT) catalyses that of the remodeling pathway. Brain PAF-PCT is insensitive to sulfhydryl requires MgZi or Mn" and it is reagents, inhibited by Ca2+. LysoPAF-AT requires Ca*' and it is inhibited by unsaturated acyl-CoA derivatives. Both enzymes are present in brain cortex, striatum, hippocampus and cerebellum. In these areas the specific activity of PAF-PCT is always more elevated than that of lysoPAF-AT. PAF-PCT activity is however particularly high in the cerebellum.
OF IN
P. W. Kiihl Institute of Theoretical Biology, Schaullstr. 2, CH-4142 Miinchenstein BL, Switzerland A large body of experimental data sugagonist bind reCeptOrS that many gests molecules not only at the "active centre" but also at one or more "peripheral sites". To the latter either an allosteric regulatory function or (in some cases) an association rate enhancing function has been attributed. We suggest another possibility: peripheral sites by which the rooms may serve a* waiting arrivals of agonist molecules at the active centre can be temporally adjusted to the inherent working rhythm of the receptor. A change of the temporal pattern of arrival as it events (e.g. from random to periodic, may occur after passage through a waiting room) may affect the working efficiency of a receptor either positively or negatively receptor's depending on where within the working cycle the arrival events are placed. The waiting-room concept is equally well applicable to enzymes with multiple substratebinding sites and is basically different from conventional allosteric concepts. 84
REGULATION METABOLISM MEDIATED CORTEX _
OF
PHOSPBATIDYLSERINE A G-PROTEIN MECHANISM IN RAT BRAIN BY
R. Mozzi, V. Andreoli, S. Pelli, Instituto di Biochimica e Chimica Medica, Universita di Perugia, Perugia, Italy. Transmembrane signal I.ransductiorr for d varirry 0f neurotransmitters in the central nerwii~ system involves a phospholipaseC-mediated hydrolysis of PlPZ with
the
production
of
IP3
and
diacylglycerols.
Tllc-
Latter compound activates protein kinase C. As in other cellular types, several reports stronqly suggest that phospholnositidephospholipase C is activated by
B C-protein mediated mechanism. The activiiy or PKC is str1ctiy dependent on prescnir> <;f the (PS) which is synthesized :n phosphatidylserine mamm?.lian tissues by base-exchange. demonstrated that PS synthesis experimental tnat newly transduction also
PS
we in
have recen:~y increased in and suggested
hypoxia in rat brain cortex synthesized PS might affect mechanisms. We have investigated
metabolism
incubating rat :.or:centraLions
is
regulated
by
signal whether
G-protein
by
brain cortical homogenate with varicus of fluoroaluminate (1-10 ml.9 NapI or
c21
81
TRANSFER OF BETWEEN MICROSOMBS IN RAT BRAIN
of the ESN
82 PHOSPHATIDYLSERINE AND MITOCHONDRIA
0. Arienti, E. Carlini and L. Corazzi Istituto di Biochimica e Chimica Medica, de1 Giochetto, 06100 PERUGIA (Italy)
ON TEE FUNCTIONAL SIGNIFICANCE MULTIPLE AGONIST-BINDING SITES RECEPTORS. Via
Phosphatidylscrine in (PS) is synthesized brain microsomes through base-exchange. Yet, its decarboxylation to phosphatidylethanolamine (PE) occurs on the inner mitochonof the transdrial membrane. The mechanism fer from endoplasmic reticulum to mitochondria is still a debated question. Indeed, it has been ascribed to energy-dependent systems, to collision, or to other mechanisms. To characterize the transport of PS between rat brain subcellular fractions, we reconstituted in vitro a system able to synthesize and dacarboxylate PS. Brain homogenates subcellular and fractions were prepared using standard procedures and incubated in the presence of Caa+ and radioactive serine (base-exchange conditions) or in a medium free of Cd+ (PS decarboxylase conditions).
Rat brain homogenates could synthesize and decarboxylate PS; the same was true for a mixture composed of purified mitochondria and microsomes, that no soluble indicating factors were necessary for the transfer and bearing out a collision-based transport. The PS newly synthesized by base exchange was preferentially transferred, infeered examininp the dioactivities of micrcsomal
as can be specific raand mitochon-
drisl lipids. 83 PLATELET-ACTIVATING FACTOR (PAF) SYNTHESIZING RAT ENZYMES IN CENTRAL SYSTEM: NERVOUS PROPERTIES AND DISTRIBUTION. E. Francescangeli, A. Boila and G. Goracci. Institute di Biochimica e Chimica Medica, Universita di Perugia, Perugia, Italy. PAF is a lipid mediator which is present in the nervous tissue and is involved in physiological functions and in pathological situations (Goracci, 1990). TWO metabolic routes have been described for the synthesis of PAF in a variety of tissues and cell the de types: IlO”0 and the pathway remodeling pathway. Previously, we have demonstrated that rat br;ain may synthesize PAF by both pathways (Francescangeli and 1989; Goracci and Francescangeli, Goracci, 1991). PAF-synthesizing phosphocholinetransferase (PAF-PCT) catalyzes the last reaction of the de nova synthesis whereas lyso-PAF acetyltransferase (LysoPAF-AT) catalyses that of the remodeling pathway. Brain PAF-PCT is insensitive to sulfhydryl requires MgZi or Mn" and it is reagents, inhibited by Ca2+. LysoPAF-AT requires Ca*' and it is inhibited by unsaturated acyl-CoA derivatives. Both enzymes are present in brain cortex, striatum, hippocampus and cerebellum. In these areas the specific activity of PAF-PCT is always more elevated than that of lysoPAF-AT. PAF-PCT activity is however particularly high in the cerebellum.
OF IN
P. W. Kiihl Institute of Theoretical Biology, Schaullstr. 2, CH-4142 Miinchenstein BL, Switzerland A large body of experimental data sugagonist bind reCeptOrS that many gests molecules not only at the "active centre" but also at one or more "peripheral sites". To the latter either an allosteric regulatory function or (in some cases) an association rate enhancing function has been attributed. We suggest another possibility: peripheral sites by which the rooms may serve a* waiting arrivals of agonist molecules at the active centre can be temporally adjusted to the inherent working rhythm of the receptor. A change of the temporal pattern of arrival as it events (e.g. from random to periodic, may occur after passage through a waiting room) may affect the working efficiency of a receptor either positively or negatively receptor's depending on where within the working cycle the arrival events are placed. The waiting-room concept is equally well applicable to enzymes with multiple substratebinding sites and is basically different from conventional allosteric concepts. 84
REGULATION METABOLISM MEDIATED CORTEX _
OF
PHOSPBATIDYLSERINE A G-PROTEIN MECHANISM IN RAT BRAIN BY
R. Mozzi, V. Andreoli, S. Pelli, Instituto di Biochimica e Chimica Medica, Universita di Perugia, Perugia, Italy. Transmembrane signal I.ransductiorr for d varirry 0f neurotransmitters in the central nerwii~ system involves a phospholipaseC-mediated hydrolysis of PlPZ with
the
production
of
IP3
and
diacylglycerols.
Tllc-
Latter compound activates protein kinase C. As in other cellular types, several reports stronqly suggest that phospholnositidephospholipase C is activated by
B C-protein mediated mechanism. The activiiy or PKC is str1ctiy dependent on prescnir> <;f the (PS) which is synthesized :n phosphatidylserine mamm?.lian tissues by base-exchange. demonstrated that PS synthesis experimental tnat newly transduction also
PS
we in
have recen:~y increased in and suggested
hypoxia in rat brain cortex synthesized PS might affect mechanisms. We have investigated
metabolism
incubating rat :.or:centraLions
is
regulated
by
signal whether
G-protein
by
brain cortical homogenate with varicus of fluoroaluminate (1-10 ml.9 NapI or