Creatine and Creatinine Metabolism - Physiological Reviews

July 2000 CREATINE AND CREATININE METABOLISM 1123
the inhibitory constant (K i) of AGAT for ornithine is �250
�M (897). Interestingly, such inhibition of AGAT by 10- to
20-fold increased concentrations of ornithine most probably
is the underlying basis for the decreased levels of
tissue Cr in patients with gyrate atrophy of the choroid
and retina (see sect. IXA) (897, 899). In isolated tubules of
the rat kidney, guanidinoacetate synthesis was shown to
be suppressed, besides ornithine, by DL-norvaline and Met
(975).
In accordance with the postulate that a sulfhydryl
group of AGAT is involved in catalysis, sulfhydryl reagents
like p-chloromercuribenzoate, DTNB, 2,4-dinitrofluorobenzene
(DNFB), and Cu 2� inactivate the enzyme
(for reviews, see Refs. 634, 1077). Purified human
AGAT is also inhibited by Hg 2� ,Zn 2� , and Ni 2� (388).
Finally, even purified preparations of hog kidney AGAT
display hydrolytic activity amounting to �1% of the
transamidinase activity (134). In contrast to the transamidinase
activity, the hydrolytic activity of AGAT is not
affected by sulfhydryl reagents.
B. S-Adenosyl-L-methionine:N-guanidinoacetate
Methyltransferase
In vertebrates, the highest levels of GAMT are found
in liver, and it has been estimated that the amount of Cr
synthesized in this organ is sufficient to meet the requirements
for Cr of the entire animal (1130; for reviews see
Refs. 1056, 1077). Intermediate levels of GAMT were detected
in mammalian pancreas, testis, and kidney,
whereas the specific GAMT activity in spleen, skeletal and
cardiac muscle, mouse neuroblastoma cells, and human
fetal lung fibroblasts was reported to be rather low (149,
664, 1056, 1077, 1129, 1130, 1135, 1136). It is not yet
known to what extent all these tissues contribute to total
in vivo Cr biosynthesis. However, because the specific
GAMT activity is on the order of 0.2–0.3
nmol � min �1 � (mg protein) �1 in liver, but only �0.005 up
to maximally 0.014 nmol � min �1 � (mg protein) �1 in heart,
skeletal muscle, fibroblasts, and neuroblastoma cells
(37°C, pH 7.4–8.0) (149, 713, 1135, 1136), the estimation
of Daly (149) that these latter tissues contain 5–20% of the
specific GAMT activity of liver seems high. The overestimation
may be explained by the facts that 1) in the study
of Daly, hepatoma cells instead of authentic liver tissue
were chosen as reference and 2) GAMT activity had previously
been shown to decrease gradually with the progression
of hepatocarcinoma (1019, 1136). Nevertheless,
at physiological extracellular concentrations of guanidinoacetate
and Cr (25 �M each), cultured mouse neuroblastoma
cells synthesized as much Cr as they accumulated
from the medium (149). In the liver and pancreas of
alloxan-diabetic rats and sheep, respectively, GAMT activity
was shown to be decreased by 50–70% (354, 1128).
In thorough studies on the tissue distribution of
GAMT expression in the mouse and rat by Northern and
Western blotting (543, 545), high levels of GAMT mRNA
were observed in the rat in kidney, caput epididymis,
testis, brain, and liver; moderate levels in the cauda epididymis;
but low or undetectable levels in the lung, pancreas,
spleen, vas deferens, prostate, seminal vesicles,
coagulating gland, heart, skeletal muscle, and small intestine.
In the mouse, highest amounts of GAMT mRNA and
protein were detected in testis, caput epididymis, and
female liver, followed by ovary and male liver. While
barely detectable signals were observed for kidney, skeletal
muscle, heart, uterus, and oviduct, no GAMT mRNA
or protein at all was found in brain, small intestine, seminal
vesicles, lung, vas deferens, cauda epididymis, coagulating
gland, or spleen. Most striking is the difference in
GAMT expression between female and male liver, which
might indicate that liver is the principal site of Cr biosynthesis
in the female mouse, whereas testis and caput
epididymis take over at least part of this function in the
male. It must, however, be kept in mind that this finding
might be due instead to the different age of the male and
female mice studied. Immunohistochemistry demonstrated
that in mouse testis, GAMT is localized primarily
in the seminiferous tubules and, in particular, in the Sertoli
cells (see also Ref. 664). In the caput epididymis,
microvilli of epithelial cells lining the initial segment of
the epididymal tubule were most intensely stained. In
contrast, spermatocytes, spermatids, cauda epididymis,
the stroma of the epididymis, and seminal vesicles displayed
no specific signals (545; see also Ref. 693). The fact
that seminal vesicles of the mouse and rat nevertheless
contain considerable quantities of Cr and PCr seems to be
due to Cr uptake from the blood via the Cr transporter
(543).
Purified GAMT from rat and pig liver is a monomeric
protein with a M r of 26,000–31,000 (397, 713). This has
been corroborated by cDNA and gene sequencing, showing
that rat, mouse, and human GAMT are 236-amino acid
polypeptides with a calculated M r of �26,000 (409, 427,
712). The human GAMT gene has a size of �5 kb, contains,
like the rat and mouse GAMT genes, six exons, and
was mapped to chromosome 19p13.3 that is homologous
to a region on mouse chromosome 10 containing the
jittery locus (114, 427). Although jittery mice share some
of the neurological symptoms of children suffering from
GAMT deficiency (see sect. IXG), sequencing of the GAMT
gene of jittery mice revealed no mutations in the coding
regions (427). Cayman-type cerebellar ataxia, a human
genetic disorder exhibiting some overlapping symptoms
with GAMT deficiency, has also been mapped to human
chromosome 19p13.3 (see Ref. 114).
In contrast to the recombinant protein expressed in
E. coli, native rat liver GAMT is NH 2-terminally blocked,
with no influence of this modification on the kinetic prop-