1
Polyamines
An Introduction
David M. L. Morgan
1. Introduction
Although receiving little attention m biochemical and phystological text-
books, the polyammes have a long history and have accumulated a constder-
able literature (for reviews see refs. l-21) In 1678 Antoni van Leeuwenhoek
(22) described crystals that formed in samples of human semen that had been
left to cool. It IS now clear that these crystals were spermme phosphate The
phenomenon was rediscovered several times durmg the next 200 years, m
each case the discoverer apparently unaware of what had gone before. By the
end of the 19th century there were ten different names for these crystals.
The structures of the three most commonly occurrmg natural polyammes,
putrescine, spermidme, and spermine, together with those of 1,3-diammopropane
and cadaverme (1,5-dtammopentane), are shown m Fig. 1. One or more of
these compounds are present m every hvmg cell. All have been found m
eukaryotes, but spermme rarely occurs in prokaryotes. In addttion to spermme,
spermidme, and putrescme, a large number of other linear, and some branched-
chain, polyamines have been detected in mammalian tissues and excreta, or
m plants, bacteria, and microorgamsms (13-16) (Table 1). The polymer-hke
nature of these compounds has led to an abbreviated way of denoting their
structures. Because the molecule starts with a primary ammo group (H,N-)
linked to a chain of methylene groups (-CH2-) interspersed with secondary
amino groups (-NH-), and terminates with another primary ammo group (-NH,);
the structure can be described simply by denoting the number of carbons
between each ammo group. Thus putrescine becomes 4, spermidme 3-4, sper-
mine 3-4-3, and so on (Fig. 1 and Table 1). Branched-chains are denoted by
From Methods m Molecular Bology, Vol 79 Polyamme Protocols
Edlted by D Morgan Humana Press Inc , Totowa, NJ
3

4
Dlaminopropane
(3)
Morgan
Putresctne
(4)
2
Hz 1
-NH*
Cadaverlne
(5)
Spermldme
(3-4)
H
Spermine
(3-4-3)
Fig 1 Structures of putrescme, spermldme, and spermme, together with dlammo-
propane and cadaverme. Frgures in parentheses below the name correspond to the
numbering system described m Subheading 1. and are derived from the smaller
figures above each bond
enclosmg the side-chain m brackets and placing It next to the atom to which rt
is attached. The N-acetyl polyammes (Fig. 2) are the most common naturally
occurrmg derivatrves m humans; however, many other polyamme metabohtes
have also been identified (Table 2).
Polyammes appear to be constttuents of many compounds found in plants and
msects. N-Carbamoylputrescme (Fig. 3) and coqugates of hydroxycmnamoyl
putrescine, and caffeoylputrescme (Fig. 3) are among polyamine mtermedr-
ates that have been isolated from plant trssues. N-Methylputrescme is a precur-
sor of the tropane alkaloids (2 7,18) and putrescine-containing alkaloids have
also been Isolated from the mar-me gastropod mollusk Monodonta labzo (19)

Table 1
Some of the Less Common Polyamines, and the Short Notation for Each Structure=
Name Chemical formula
Abbrevtatton
Dtaminopropane NHACWW,
3
Putrescine NH,K%hNH,
4
Cadaverme
W(CH,),NH,
5
Norspermtdme NH2WWWCH2),NH,
3-3
Spermidine
NH2W,WWb),NH,
3-4
Ammopropylcadaverme NH,(CH,),NWCW,NH,
3-5
Homospermrdme NH2(CH,)NTCWJW
4-4
Norspermme
NH2(CH,),NH(CH,)sNH(CH,),NH,
3-3-3
Thermospermme NH,(CH,),NH(CH,),NH(CH,),NH,
3-3-4
Ammopentylnorspermtdme NH,(CH,),NH(CH,bNH(CH,),NH,
3-3-5
Spermine
NH,(CH,),NH(CH,),NH(CH,),NH,
3-4-3

Brs(ammopropyl)cadaverme NH,(CH,),NH(CH,),NH(CH,),NH2
3-5-3
Ammopropylhomospermme NH2(CH,),NH(CH,),NH(cH,),NH,
3-4-4
ol
Canavalmme
NH,(CH2)4NH(CH,),NH(CH,),NH,
4-3-4
Homospermme NH2(CH,),NH(CH,),NH(cH,),NH,
4-4-4
Caldopentamme NH2(CH,),NH(CH2)3NH(CH2hNH(CH2)3NH,
3-3-3-3
Ammopropylcanavalmme
NH2(CH2)3NH(CH2)4NH(CH,)3NH(CH2)4NH, 3-4-3-4
Bu(ammopropyl)homospermrdme NH2(CH,),NH(CH,),NH(CH,),NH(CH,),NH,
3-4-4-3
Bts(ammobutyl)norspermtdme
NH,(CH2)4NH(CH2)3NH(CH2hNH(CH2)4NH,
4-3-3-4
Ammobutylcanavalmme
NH,(CH,),NH(CH,)3NH(CH,),NH(CH,),NH,
4-3-4-4
Ammopropylhomospermme NH,(CH,),NH(CH,),NH(CH,),NH(CH,),NH,
3-4-4-4
Homopentamme
NH2(CH,),NH(CH,),NH(CH,),NH(CH,),NH, 4-4-4-4
p-ammobutylhomospermme
NH2(CH,>,N((CH,),NH,)o,NH(CH,),NH,
4(4)-4-4
Caldohexamme

NH,(CH,),NH(CH,)3NH(CH,),NH(CH2)3NH,(CH,)3NH2
3-3-3-3-3
Homocaldohexamme NH,(CH,),NH(CH,),NH(CH,),NHo,NH,(CH,H2
3-3-3-3-4
Thermohexamme NH2(CH,),NH(CH,>,NH(CH,),NH(CH,),NH,(CH,),NH,
3-3-3-4-3
Homothermohexamme NH2(CH,),NH(CH,),NH(CH2)4NH(CH2)3NH,(CH2)3NH2
3-3-4-3-3
Agmatme
I@-methylagmatme
NH2C(NH)NH(CH2)4NH2
NH,C(NCH3)NH(CH,),NH,
OAlso a number of branched-cham penta-, hexa-, and hepta-ammes (4(4)-4-4-4, 4-4(4)-4-4-4, 4-4(4)-4-4, 4(4)-4(4)-4, and so on) have
been found m plant seeds Boldface indrcates the three most common polyammes
6
CH,Co NH2
H
Morgan
N-Acetylputrescme
CHQ-~N-NH2
H H
Hz-N-
H
@-Acatylspermldme
CH~CC+N ~“J~~~
H H
ti-Acetylspermme
CHICO ~
LOCH,
H H

N1 ,NJ2-Diacetylspermine
Fig 2 Structures of the common N-acetyl polyammes (the acetyl groups are m
bold type) The numbermg system used to mdlcate the powon of the acetyl group 1s
that of Tabor (23)
Palustrine, maytenine, and cannabisatrvme (from maquana) are examples of
the many spermldine-containing alkaloids found m plants (Fig. 3) (20)
Aphelandrme IS a spermine-containing alkaloid from the flowermg shrub
Aphelandra tetragona.
Low-mol-wt spader and wasp toxins, whrch are selec-
tive inhibitors of glutamate receptors of the central nervous system (CNS),
consist of a polyamine backbone to which are linked one or several carboxylic
acids and/or amino actds
(Fig.
3) (21). Hydroxylamme-containmg polyammes
have been isolated from the venom of the funnel-web spider
Agelenopszs aperta
(22). However, in many cases tt 1s not clear whether the polyamines are precur-
sors m the biosynthesis of these compounds. A glutathronyl-spermidine coqu-
gate, N’-monoglutathionyspermidme IS found m
Escherichza coli;
trypanothrone,
Polyamiff es
7
Table 2
Some Polvamine Metabolites Identified in Human and Rat Urine (24
Diammopropane
N-Acetyldlaminopropane
P-Alanmen
y-Ammobutyrrc acIda
Cadaverine

N-Acetylcadaverme
&Ammovaleric acid
Isoputreanme (N-(3-ammopropyl)-4-ammobutync acid
Putreanrne (N-(4-ammobutyl)-3-ammopropiomc acid
Spermldlc acid (N-(2-carboxyethyl)-4-ammobutync acid
Spermlc acid- 1 N-(3-ammopropyl)-N’(-(2-carboxyethyl)- 1,4-dlammobutane)
Spermic acid-2 (N,N-brs(2-carboxyethyl)-1,4-diaminobutane)
%an also arise from nonpolyamme sources Polyamme ongin of these metabohtes
was confirmed by the use of isotoplcally labeled precursors
a glutathlone-spermldme conjugate
(Fig. 4),
is apparently unique to trypano-
somes, where it appears to substitute for glutathlone (25). Castanospermine (26),
aspldospermidine, and aspldospermme (27,28), are
not
polyamme derivatives.
The polyamines are, m the main, lmear ahphatlc molecules of, m biological
terms, small molecular mass. They are water soluble, and at physiological pH
all the ammo groups will be positively charged; hence, these compounds are
organic bases, their baslclty increasing with the number of ammo groups.
Unlike inorganic molecules or ions, the positive charges on polyamines are
spaced out at intervals and, although the hydrocarbon chains are flexible, will
have sterlc as well as catiomc properties.
The difficulties experienced m attempting to identify and measure the
amounts of polyammes m biological materials follow from three characteris-
tics: their size, the low concentrations in which they are present, and that their
only reactive centers are the ammo groups. Thus, methods for polyamine
analysis must include procedures to extract, and perhaps concentrate, the
polyammes; to separate them from other ammo-containing compounds (such
as amino acids), and from each other; to convert them into colored or fluores-

cent derivatives; to identify them; and then to make quantitative measurements.
Almost all of the methods available to analytical biochemists have been applied
to polyamine analysis at some time (29,M) (see Chapters 13-l 6).
2. Biosynthesis (see Chapters 2-5)
With the exception of laboratory-bred mutant cell lines, all cells have the
ability to synthesis putrescme and spermidine. Spermme synthesis appears to
8
Morgan
H
F
P
H* H
G
Ftg 3. Examples of polyamme-containmg natural compounds N-carbamoyl-
putrescme (A), 4-coumaroylputrescme (B), caffeoylputrescine (C), the spermtdme
alkalotds cannabrsattvme (D), palustrme (E), and maytenme (F), the spader toxin
NSTX-3 contams both putrescme and cadaverine residues (G). The emphasized bonds
indicate the polyamine moreties
be confined to eukaryotic systems. There are, however, differences of detail
between the pathways in different cell types, and a generalized pathway of
polyamme btosynthesrs 1s shown m Fig. 5. In mammalian cells and m fungt the
initial, and at this stage rate-lrmitmg, step 1s the decarboxylation of ornithtne to
form putrescme, catalyzed by ormthine decarboxylase. The source of the start-
ing material (orntthme) 1s not entirely clear. In animals ornithine 1s present in
Polyamlnes
coo
H3+cpNx;
2
+H
CH2)4

SH
0 /
I
NH
Trypanothlone
Fig 4 The unrque glutathione-spernudme conJugate of trypanosomes (the spernn-
dine moiety is in bold type).
the blood plasma (human plasma contams about 85 l-m-101/L /31/), some of which
will be dietary m origm. Omithme is also a product of the urea cycle, and tt
seems probable that some may be diverted to polyamine biosynthests. Further-
more, many cells that lack a complete urea cycle contam argmase, and the pres-
ence of thus enzyme will ensure the availabihty of ormthine for polyamine
production. Thus, m these cells hydrolytic cleavage of the guamdmo group of
argmine may be the fn-st step m polyamme biosynthesis (32). In mammahan
cells and fungi there is only one pathway for putrescme synthesis, but many
micro-organisms (33) and higher plants (34,35) possess a second constitutive
pathway vra agmatine, itself formed by the decarboxylatton of argmme by argl-
nme decarboxylase. Agmatme 1s then hydrolyzed by agmatmase (agmatme
amidinohydrolase) to form putrescine, with the ehmmatton of urea (Fig. 5). Some
organisms, e.g ,
E
colz, possess both of these pathways. In plants addmonal routes
exist from agmatme to putrescme; agmatme immohydrolase yields ammonia
and N-carbamoylputrescme, which 1s hydrolyzed by N-carbamoylputrescme-
amidohydrolase to give ammoma, carbon dioxide, and putrescme (Fig. 5). It is
intriguing that agmatme has recently been found m rat ttssues (36).
In a step common to most orgamsms, spermrdme 1s formed from putrescine
by addition of an ammopropyl group donated by decarboxylated S-adenosyl-
methtonme, a reaction catalyzed by spermidme synthase, an ammopropyl-
transferase. Addition of a second ammopropyl moiety to spermtdme, catalyzed

by a different ammopropyl transferase, spermine synthase, forms spermme
(37). The source of the ammopropyl group 1s a second molecule of decarboxy-
lated S-adenosylmethtomne. The synthesis of spermtdme and spermme IS
10 Morgan
3-Acetamdopropanal
0acarboxylatad
S-Adenosylmethwmne
N1 -Acetylspermidine
ZLAcetamdopfopanal
DWXbX#bd
S-Adervxyimethwtne
S-Adenosyi-
meltucwte
Fig. 5. The mam pathways of polyamme blosynthesls m ammals, plants, and
microorganisms The enzymes involved are (1) argmase (EC 3 5 3 l), (2) ormthme
decarboxylase (EC 4.1 1 17); (3) argmlne decarboxylase (EC 4 1.1 19); (4)
agmatinase (EC 3 5.3 11); (5) agmatme delmmase (EC 3.5.3.12), (6) N-carbamoyl-
putrescme amldase (EC 3 5.1 53); (7) spermidme synthase (EC 2 5 1 16), (8) S-adeno-
sylmethlonme decarboxylase (EC 4.1.1.50), (9) spermme synthase (EC 2.5 1 22),
(10) spermldme/spermine N1-acetyltransferase (EC 2 3 1 57); (11) polyamme 0x1-
dase(EC 15 3.11)
iJoiyam/nes
11
dependent on the availability of the aminopropyl donor, hence S-adeno-
sylmethionme decarboxylase is also rate-limitmg m polyamme biosynthesis.
The methionme and adenosine moieties of 5’-methylthioadenosine, the other
product of the ammopropyltransferase reactions, are salvaged by a series of
reactions that differ between mammahan cells on the one hand, and plants and
bacteria on the other, and are not yet fully understood (9). In a number of bac-
terra, and m some plants, L-asparttc+-semialdehyde (38) is the ammopropyl

group donor m spermidme biosynthesis.
The three key enzymes that regulate polyamme biosynthesis are ormthine
decarboxylase, S-adenosylmethionine decarboxylase, and spermidine/spermine
N’-acetyltransferase (39), the enzyme that mitrates polyamine catabolism;
the activrtres of the other enzymes m the pathway shown m
Fig. 5
appear to be
governed by the avarlabihty of the appropriate substrate. Each of these enzymes
wtll now be discussed in more detail.
2.1. Ornithine Decarboxylase (E. C.4.1.1.17; see Chapters 2 and 3)
Ornithme decarboxylase (ODC) has received more attention than any other
enzyme of the polyamme btosynthetic pathway (40-M). The enzyme proved
difficult to purtfy because it 1s extremely labile, the cellular content IS very
low, and it may be present in multiple forms. However, cDNA clones containing
the codmg region for ODC have been obtamed from a number of species (43).
All forms of ODC constst of identical subunits and the complete enzyme is
generally a dimer. The subunit size of the sequenced enzymes is, m the main,
remarkably similar, except for the bacterial and Leishmanza enzymes, which
are about 40% bagger. There are also remarkable similarities m the sequences
of the eukaryotic enzymes, with more than 90% identity between the mamma-
lian enzymes. Even Leishmania ODC shares 40% identity with mouse ODC
over the core region In contrast, there is very little similarrty between the
sequences for the bacterial enzymes (from E cob) and those from eukaryottc
species. The close identity between the ammo acid sequences of the mamma-
lian enzymes suggests that their structural and catalytic properties are also very
similar. E. colz appears to be unique in that some strains contain two different
forms of ODC that are mmmnologrcally distinct, a biosynthetic (constitutive)
enzyme that resembles those of other organisms and a biodegradative form
that may use available omrthme as a carbon source (33,451.
ODC, from whatever source (Including plants), has an absolute reqmrement

for pyridoxal 5’-phosphate for activity. The binding site for pyrrdoxal phos-
phate is at lysme-69 m mouse ODC (46), which 1s in a highly conserved region
within the eukaryotic enzymes. In contrast, the sequence containing the bmd-
ing site in E. coli ODC IS completely
different (43). Many forms of the enzyme
require the presence of sulfhydryl reducing agents (such as dithrothreitol) for
12 Morgan
maximal activity and this may well be because of the presence of a cysteme
residue in the pyridoxal phosphate bmdmg site.
ODC protein has an extremely short half-life, of the order of lo-60 min,
depending on species. Deletion of some or all of the 36 residues at the carboxyl
end of the mouse enzyme does not alter enzyme activity, but greatly increases
the stability of the protein and prevents its rapid turnover (47,481. The carboxy
terminal part of mouse ODC contains a PEST region (rich m prohne, glutamic
acid, serme, and threonme; residues
423449) (49). Trypanosoma
ODC does
not have a carboxyl PEST sequence and is not rapidly degraded, but this stab&
ity was reduced when
Trypanosoma brucez
ODC expressed m Chinese ham-
ster ovary cell had an added mouse ornithme decarboxylase C-termmus (SO).
However,
Xenopus
ODC does not have a clear carboxy PEST region, yet has a
rapid turnover.
The sole function of ODC appears to be to catalyze the decarboxylation of
ornithme, an ammo acid not incorporated mto proteins, to form putrescine, the
nntial diamme m the polyamine biosynthetic pathway, 90% of the activity is
cytoplasmic. In normal cells ormthme decarboxylase activity is very low and it

has been estimated that there may be only 100-200 molecules of enzyme in a
quiescent cell (51).
2.2.
S-Adenosylmefhionine
Decarboxykse
(E.C. 4.1.1.50; see Chapter 4)
S-Adenosylmethionme decarboxylase (AdoMetDC) the second of the rate-
limiting enzymes in polyamme biosynthesis, IS essential for the formation of
spermidme and spermidme m mammalian cells (52,53), plants (35,54), m
micro-organisms such as
E colz, Saccharomyces cerevlsiae, Physarum
polycephalum, Neurospora crassa,
and
Asperglllus nldulans
(33) and prob-
ably m all organisms that synthesize these polyammes. All forms of AdoMetDC
examined so far are synthesized as a proenzyme. cDNA clones coding for the
proenzyme have been isolated from
E colz,
S
cerevuzae,
human, bovine, rat,
and hamster tissues (see ref. 53). Mammalian AdoMetDC is synthesized as a
proenzyme of about 38,000 molecular mass, which is cleaved autocatalytically
to form two unequal sized subunits of about 31,000 (a-) and 7000 (p-). The
cleavage occurs between glutamate-67 and serine-68; the glutamate becoming
the carboxy termmus of the P-subunit whereas the serme becomes the ammo
terminus of the a-subunit Both subunits are necessary for catalytic activity,
the proenzyme is inactive. The ammo-acid sequence of the protein is highly
conserved among different mammahan species with about 90% identity among

human, rat, bovine, and hamster enzymes.
All known forms of AdoMetDC contain covalently bound pyruvate. On
cleavage of the proenzyme, the ammo terminal serme of the a-subunit is con-
Polyamines
13
verted to the pyruvate prosthetic group. In AdoMetDC the carbonyl group of
pyruvate IS used to form a Schiff base with the substrate, m contrast to the use
of pyridoxal phosphate m ormthme decarboxylase.
AdoMetDC is an essential component of the pathway for spernudine and
spermme biosynthesis. Decarboxylatton of S-adenosylmethionine commits this
compound to the polyamme pathway and prevents it acting in its usual role as
a methyl donor. The active site of AdoMetDC appears to contain a cysteme
residue that IS essential for enzyme activity, but does not form part of a disul-
fide bridge. This may explain the stimulatory effect of reducing agents, such as
dithiothreitol, on enzyme activity. AdoMetDC has a short half-life of the order
of 1 h or less m mammalian cells (53). There is a PEST region m the a-subunit,
but whether this has an effect on the stability of the enzyme has not been estab-
lished. Breakdown is enhanced m the presence of spermidme or spermine, but
not putrescine
2.3. Spermidine Synthase (E.C. 2.5-l. 76)
and Spermine Synthase (E.C. 2.5.1.22; see Chapter 5)
Spermidme synthase and spermine synthase are constitutively expressed
aminopropyltransferases that are much more stable than either ODC or
AdoMetDC. They catalyze the transfer of an ammopropyl group from decar-
boxylated S-adenosylmethionme to putrescme or spermidme; the activities of
these synthases are regulated by the availabihty of their substrates. As a conse-
quence of their lack of a regulatory, or rate-limiting, role m polyamme biosyn-
thesis, these enzymes have been less studied than the two decarboxylases.
Spermidme synthase has been isolated and characterized from bacterial (55),
plant (56), and mammalian (5741) sources. In each case the enzyme consists

of two identical subunits of equal size. Spermidine synthase is inhibited by
i’V-ethylmaleimide and p-hydroxymercuribenzoate and is stimulated by
dithiothreitol or 2-mercaptoethanol. Hence, m common wrth the two decar-
boxylases, this appears to be a sulfhydryl-requiring enzyme. Cadaverme (1,5-
diaminopropane) and spermidine can also act as substrates for the mammahan
enzyme, but the reaction proceeds at only l/20 of the rate with putrescme.
Spermidme synthase from bovine brain can also utilize 1,6-diaminohexane as
an aminopropyl receptor but only at 1% of the rate with putrescme. Both the
reaction products, spermidme and 5’-methylthioadenosine, will mhibit the
enzyme. Human spermidme synthase has been cloned (62) and the amino-acid
sequence deduced from the cDNA.
Spermine synthase has been isolated from bovine (58) and human tissues (61).
Both forms of the enzyme, like the spermidine synthase, consisted of two sub-
umts of equal size. Both were inhibited by the reaction products, spermine and
S-methylthioadenosme. Putrescme is a competitive inhibitor of spermidme for
the bovine enzyme Spermme synthase is inhibited by N-ethylmaleimide and
p-hydroxymercuribenzoate, and this mhibition can be restored by 2-mercapto-
ethanol or dtthiothrenol.
3. Polyamine Catabolism (see Chapters 6-12)
In contrast to the extensive studies of polyamme biosynthesis, polyamme
degradation has received much less attention, possibly because it offers fewer
possibihties for control of cellular activrty. Polyammes are oxidized m plants,
bacteria, fungi, and animals by a variety of oxidases with differing modes of
action and cofactor requirements. In mammahan cells spermine and spernndme
can be converted back to putrescine by the pathways shown m
Fig. 5.
The first
step IS the acetylation of one of the two ammopropyl groups of spermme, catalyzed
by the enzyme spermidme/spermme N’-acetyltransferase, to give N’-acetyl-
spermme. This m turn is degraded by a polyamme oxidase with the formation

of spermidine and an aldehyde, 3-acetamldopropanal The smgle aminopropyl
group of spermidme is also acetylated by spermidme/spermme Nr-acetyl-
transferase and the resulting N’-acetylspermidme can be cleaved by a
polyamme oxidase to form putrescine and 3-acetamidopropanal. The putrescme
can be either recycled to sperrmdme and possibly spermme
(Fig. 5)
or further
metabolized.
3.1. Spermidine/Spermine N’-Acetyltransferase
(E.C. 2.3.1.57; Diamine N-Acetyltransferase; see Chapter 6)
Spermidme/spermme N1-acetyltransferase (M-SAT), which appears to be
ubiquitous m mammalian tissues (39) catalyzes the transfer of an acetyl group
from acetyl-coenzyme A to an ammopropyl moiety of spermme or spermidme
(Fig.
5). The N’-acetylspermme or N’-acetylspermidme is then oxidized by the
constitutive intracellular polyamme oxidase, which cleaves the polyamine at a
secondary amino nitrogen to release 3-acetamrd ammopropanal (N-acetylammo-
propionaldehyde)
(Fig.
6). Tissue polyamme oxidase activity is usually sufficient
to ensure that mtracellular levels of Nr-acetylsperrmdme or N’-acetylspermme
are below the limits of detection, thus the rate of polyamme degradation is
regulated by the activity of N’SAT. It IS not present m plants, but a similar
enzyme has been found m E coli (33). Candlda boldinil (63) contams an
acetyltransferase that forms both N’- and N8-acetylspermidme in approximately
equal proportions, and will also acetylate putrescine and diaminopropane. @SAT
has been purified from rat, human, hamster, and chicken tissues (for references
see
ref.
39). The purified enzyme is unstable and IS particularly sensitive to

heat. It is not clear whether spermrdine or spermine is the preferred substrate m
viva; both polyamines are acetylated m cells m which the enzyme has
been activated N’-SAT acetylates the ammopropyl end of spermidme, only
Polyamines
75
L 4
H2N(CH2hNH(CH2)4NH(CH&NH2 spermine
OCH(CH,kNH(CH&NH(CH&NH2 OCH(CHzkNH(CH2),NH(CH?)2CH0
Mmoaldehyde
ChIdehyde
A
H2N(CHd2CH0
H2N(CH2)4NH2(CH2)3NHz
mlnopropanal Y.
El ,a’
spermtdlne
*. & ,/
H2NhH&NH(CH&NH(CH&NHZ spermlne
J
3
E2
\
WW-hhNH2
OCH(CH&NH(CH2)3NHz
dlamlnopropane
1
HC==CH
L
\
/

WhhW
H2 ‘32
l-(3-am1nopropyl)-2-pyrroltne
B
Fig. 6. Alternative sttes of action on spermine by polyamine oxrdases from differ-
ent sources. (A) Oxidation of terminal (primary) ammo groups with the production of
ammonia by, e g., the bovine serum enzyme. The arrows indicate the bond that IS
broken. (B) Enzymes that cleave the molecule at a secondary ammo group to produce
either diammopropane (E,, e g , maize polyamme oxrdase /83/ or ammopropanal EZ,
from rat liver /84/).
N1-acetylspermrdme (Fig. 2) IS produced; A@-acetylspermidme is not formed.
However, other ammes contammg an aminopropyl group linked to a secondary
amino can also act as substrates (64); but, interestingly, I@-acetylsperrmdme 1s
not further acetylated. Wtth spermme the product is N’-acetylspermme that
can be further acetylated to form N’,Ni2-diacetylspermme (65,66), a compound
that has been detected m the urme and ammotic flurd of pregnant women (67).
N’SAT has no deacetylase activity, hence the catalytic mechanism IS essen-
tially irreverstble. Human IV’-SAT crossreacts wrth antisera raised against the
rat liver enzyme (681, indrcatmg a high degree of mnnunologrcal identity
16
Morgan
The nucleotide sequences of cDNAs have been determined for the human
(69), hamster (70), and mouse (71) enzymes. The deduced ammo acid
sequences are more than 95% homologous. Such extensive mterspectes
homology shows a high degree of evolutionary conservatton and 1s usually
taken to indicate that such proteins have important cellular functions.
3.2. Spermidine N*-Acetyltransferase
and Acetylspermidine Deacetylase (see Chapters 7 and 8)
While not part of the mam pathway of polyamme metabolism (Fig. 5), It
seems appropriate to discuss these two enzymes here. In contrast to spermine/

spermtdme N’-acetyltransferase, which 1s a cytosolic enzyme, spermidme
N8-acetyltransferases (there may be more than one) are predommantly nuclear
and can also acetylate htstones (72,73). The N1- and p-acetyltransferases are
tmmunologtcally distmct; antisera to the former havmg no effect on the latter
(74). In contrast to the N’-acetyltransferase, spermtdme N8-acetyltransferase
activity in cultured cells IS not affected by serum-sttmulated growth or addmon
of spermidine to the culture medium (75). The N8-acetylspermidme formed m the
nucleus is exported to the cytosol where the malonty IS deacetylated by a cytosohc
acetylsperrmdme deacetylase (76,771 that, contrary to earlier reports, does not
act on N*-acetylspermrdme (78). The function of spermidine N8-acetyl-
transferase 1s not clear but, by analogy with what 1s known at the cellular level,
tt seems probable that acetylation may be a means of mactivatmg nuclear sper-
mtdme and converting tt to a form that can more easily cross the nuclear mem-
brane. The deacetylase then regenerates the spermidine to become part of the
intracellular pool.
3.3. Polyamine Oxidases (see Chapters 9-12)
Polyamines are oxtdized to form ammoaldehydes by a number of enzymes,
classified variously as EC 1.4.3 4 amine oxidase (flavm-contammg); EC
1.4.3.6. amme oxtdase (copper-contaming); EC 1.4.3.10. putrescine oxtdase,
and EC 1 S.3.11 polyamine oxidase. The trivial names associated with the
first two groups by the Nomenclature Committee of the International Union of
Biochemistry (79) (monoamme oxidase, amme oxtdase, tyramme oxtdase; and
dtamme oxidase, amme oxidase, histammase, respectively) are indicative of
the confuston in this area. The putrescine oxtdase of Mzcrococcus rubens, a
diamine oxidase, contains flavm (JO), whereas the diamme oxtdase in porcme
plasma 1s a copper-containing enzyme (81). Hence, classificatton of a parttcu-
lar amine oxrdase within these groups is often difficult, particularly so m the
case of partially purified preparations. An alternative approach (5,82), 1s to
divide the polyamme oxidases mto those that act at the primary (terminal) ammo
groups of di- and polyammes and form ammonia as one of the products (Fig.

Polyamines
17
6A; Group I); and those (the majority) that act at the secondary amino group(s)
of the aminopropyl moteties of spermine or spermidme
(Fig. 6B;
Group II).
Enzymes cleavmg a polyamme at a secondary amino group would be further sub-
divided according to whether dtammopropane (Group IIa) or 3-acetamido-
propanal (aminopropionaldehyde; Group Ilb) are among the products
(Fig. 6).
However, there are also dtfficulties with this classification, because it may
prove impossible to separate putrescme- from spermidme- or spermme-oxidtz-
mg acttvities, although the latter involve attack at a secondary ammo group.
Amine oxidases able to uttlize the polyammes spermme or spermidme as
substrates will here be considered as polyamine oxidases, whether or not they
can also act on di- or mono-amines. Some of the properties of a representative
(and mcomplete!) selection of amme oxtdases are m
Table 3.
Oxrdatron taking place at the primary (terminal) ammo groups has been
named terminal catabolism by Seiler (85), because the amino aldehydes pro-
duced cannot be recycled to polyamines. In contrast, oxidatton at a secondary
amino group with the formation of putrescme or spermidme that can be
recycled back to higher polyammes is part of the mterconversion pathway m
Seller’s terminology.
3.4. I. Copper-Containing Oxidases
The bovme plasma polyamme oxidase (EC 1.4.3.6.), can be constdered the
prototypic mammalian copper-dependent amme oxidase. The enzyme acts on
spermidme and spermme to produce, respectively, an ammomonoaldehyde
[hr-(4-ammobutyl)-ammopropanal] or a dialdehyde [NM-bzs(3-propanal)- 1,
4-diammobutane], ammonia, and hydrogen peroxide

(Fig.
6) (86); molecular
oxygen is required (87). The enzyme oxidizes polyammes containing primary
amino groups, those forming part of an ammopropyl moiety bemg the more
readily attacked (88), as well as putrescine (89,90), and some primary ammes.
Although hydrogen peroxide is produced, there is no evidence of superoxide
formation (107). Reports that acrolem IS a product of the oxidatron of spermine
or spermidine by this enzyme (108-210) persist (89), despite the careful work
of Israel et al. (ZZZ), who were unable to detect acrolem formation when syn-
thetic spermme dtaldehyde, prepared by unambiguous synthesis, was allowed
to break down under physiological conditions.
3.4.2. FAD-Containrng Tissue Amine Oxidases
No detailed mechanistic studies have been made of the flavm adenine
dmucleotide (FAD)-contammg amine oxidases. Peroxisomal and/or cytoplas-
mic FAD-contammg enzymes are found m most mammalian tissues (84,112).
Rat liver polyamme oxidase (8g catalyzes the oxidation of spermme, and sper-
midine, but not putrescme. It acts on the secondary ammo groups of spermme
Table 3
A Representative Selection of Amine Oxidases
Source (23)
Subunit Inhibition by
Inhibition
(k?a)
carbonyl by SH
Cu(II) Fe( II) reagents FAD reagents
Reference
Bacteria
Mzcrococcus rubens
Fungi
Aspergillus nzger

Plants
Gluczne max (soybean)
Zea mays (maize)
z
(PAO’)
(DA02)
Avena sativa (oat)
Pzsum satzvum (pea)
Nematode
Ascarzs suum
Mammals
Bovme plasma
Bovine liver
Rat liver
Human pregnancy plasma
Human placenta
52
255 83
3
+
92.93
113 77
65
118 55
63
131 66
66 66
180 8.5 2
+
101,102

60 57
+
103
60 60
+ +
84
67
+
104
70 69 5 13
105
+
2
-
+
+
-
+
+
80.91
94,95
-
96
+
97
98
99
+
100
9 Polyamine oxldase, 2 dlamme oxldase, 3 also contams manganese Inhlbltlon by carbonyl reagents, pnmanly semlcarbazlde, IS consld-

ered to mdlcate the presence of topaqumone (206) Sulfhydryl reagents used were mamly N-ethylmalelmlde, or p-hydroxymercunbenzoate
Polyamines 19
or spermidme with 3-aminopropanal as one of the products (Fig. 6). This was
confirmed by demonstratmg the formation of N-acetyl-3-ammopropanal, N’-
acetylspermidine, and putrescme from iV1 ,N12-diacetylspermme by this enzyme
(113). This is the FAD-dependent enzyme of Fig. 5, responsible for the recy-
cling of spermme and spermidme to putrescme An enzyme with broadly semi-
lar characteristics, except that putrescine is also oxidized, is present m human
serum during pregnancy (104,114). Polyamine oxrdase activity IS high in
most tissues. It is present at levels comparable to those of spermme and sper-
midme synthases and that greatly exceed that of spermme/spermidme N’-
acetyltransferase (9). Hence, under normal condmons tissue levels of acetylated
polyamines verge on the undetectable This FAD-contammg polyamme oxi-
dase is present m all mammalian tissues and in all cultured mammalian cells
examined so far.
4. Regulation of Intracellular Polyamine Levels
Although mtracellular polyamine concentrations vary throughout the cell
cycle (115), the maximum change rarely exceeds twofold. Polyamme concen-
trations m resting fibroblasts have been calculated as ~29 @4putrescme, 159 pA4
spermidme, and 635 pJ4 spermme; transformed and tumor cells generally have
higher levels and for SV-3T3 cells the calculated concentrations are 229, 1835,
and 694 fl, respectively (226). Intracellular polyamine levels are tightly regu-
lated by mechanisms that control biosynthesis, degradation, and uptake. ODC
activity is increased by growth factors, hormones, regenerative stimuli, tumor
promoters, immunoadjuvants, and many drugs (42,117,218), in most cases this
is because of an increase m enzyme protein. Translation and turnover of ODC
protein is regulated, both upward and downward, by changes in intracellular
polyamme levels. Degradation of the enzyme protein IS enhanced by bmdmg to a
unique mhibitory protem, antizyme, which is itself induced by polyammes (119).
The supply of decarboxylated S-adenosylmethionme is highly regulated by

changes m the amount of active AdoMetDC protein within the cell resulting
from alterations in the rate of formation of active enzyme from proenzyme, in the
rate of synthesis of proenzyme, and in the rate of degradation of active enzyme,
AdoMetDC is inhibited by its product, decarboxylated S-adenosylmethionine,
and some forms are activated by putrescine, probably by an allosteric mecha-
nism mvolvmg a conformational change in the enzyme protein. Low polyamme
levels stimulate both synthesis and translation of AdoMetDC mRNA (120,121),
synthesis of AdoMetDC proenzyme is also influenced by intracellular
polyamines. Spermidme and spermme synthases are inhibited by then- prod-
ucts, spermidme and spermme, and also by S-methylthioadenosme.
Nr-SAT is normally present only at very low levels in cells. It is induced by
spermidme and spermme, and also by a number of other agents, including hor-
20 Morgan
mones, growth factors, and a variety of toxic msults (39). In common with the
other key enzymes of polyamme metabolism, ormthme decarboxylase, and
S-adenosylmethionme decarboxylase, N’-SAT 1s a short-lived enzyme that 1s
rapidly turned over and is regulated at the levels of mRNA accumulation, mes-
sage translation, and protein turnover.
Many cell types (122,123) possess an uptake system for polyammes that 1s
distmct from those for ammo acids and that, in certain circumstances, can sub-
stitute for synthesis
de
MOW In some cells polyammes are taken up by both
saturable and nonsaturable systems. Uptake by saturable systems is energy-
requiring, temperature-dependent, carrier-mediated, and operates against a sub-
stantial concentration gradient. In many cells putrescme and, often, spermidine
transport is sodium-sensrtive (the term sodium-sensitive is preferable to the
more frequently used sodium-dependent, because removal of extracellular
sodium reduces, but often does not abohsh, uptake) In contrast, spermme trans-
port in most cells is not affected by changes in extracellular sodium Transport can

be inhibited by reagents, such as N-ethylmaleimide or p-chloromercuribenzoate,
that alkylate sulfhydryl groups, and stimulated by sulfhydryl donors, such as
dithiothreitol, mdicatmg a requirement for SH-groups on the transporter(s) that
may be within, rather than on, the cell membrane. The number of carriers m the
polyamme transport system varies with cell type. In human umbthcal vem
endothelial cells there are apparently two carriers, one shared by spermme and
spermidine, and one capable of transportmg all three polyammes
(124).
In con-
trast, porcine aortic endothelial cells appear to possess three carriers, one for
each polyamme (125). Multiple carriers are not uncommon, although many
cell types appear to possess a smgle polyamine carrier (126).
The mechamsms of polyamme transport and the means by which polyamme
uptake is mduced are not clear Uptake is generally low m quiescent cells, or m
ceils that have been induced to differentiate, m contrast to cells in rapid growth,
m which uptake is enhanced. Uptake 1s also increased m response to prohfera-
tive stimuli, such as serum, growth factors, and hormones, and m cells m
which polyamme stores have been depleted. Polyamme transport appears to be
regulated by both a rapidly degraded protein mhibitor, probably antizyme, an
ODC inhibitory protein (219,127) that responds to a rise in mtracellular
polyamme levels, and a longer-lived protein, or proteins, which may be the
polyamine carrier(s).
5. Polyamine Pharmacology
Induction of ODC is a very early event in cell proliferation, the mcrease m
enzyme activity occurring before DNA or protein synthesis. This led to con-
siderable interest m the use of polyammes as tumor markers or as mdtces of
tumor therapy (41,128,129), but this proved less successful than expected (130).
Polyamines 21
Polyamine biosynthesis is also seen as a potential target in tumor chemotherapy
(8,10,131) and in other proliferattve diseases. Inhibitors have been developed

to block one of the regulatory steps in the pathway (39,43,53,131-134). Another
ongomg approach has been the development of structural analogs of the
polyammes that would be taken up by the polyamine transport system and
selectively interfere m polyamine metabolism (135). Inhibitors of polyamme
brosynthesis have been used successfully in the treatment of some cancers and
protozoan diseases, particularly African trypanosomiasis (132,136,13 7).
6. Functions of the Polyamines
What do the polyammes do? The conventional response is to say that at
present we do not know, but this has been described as “a cautious misstate-
ment” (Marton and Morris, in ref. 132, p. 79). It is clear that adequate intracel-
lular levels of polyammes are necessary for optimal growth and rephcatton of
plant, bacteria, fungi, and, indeed, all cell types examined so far. Intracellular
concentrations of polyammes are m the high micromolar range and at physi-
ological pH all then ammo groups ~111 be positively charged. Hence, the
majority of the polyammes will be sequestered m some way and it is probable
that only the “free” polyamine pool is physiologically active. Attempts to
localize intracellular polyamines have been mconclusive (138) and, as yet, no
polyamme stores have been identified. The first unequivocally established
function for polyammes at the molecular level is the donation of a 4-aminobutyl
moiety by spermidme to the eukaryotic initiation factor 5A (eIF-5A) precursor
protein to form the ammo acid hypusine (139). Polyammes can influence the
transcriptional and translational stages of protein synthesis (see ref. 9), stabilize
membranes (l#O), alter intracellular free calcium levels (141-143), and may well
have important messenger functions. Polyammes also have important neuro-
physiological functrons (146146). To quote Coffino (143, “Polyammes are domg
some important things, but we do not know what they are ”
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