Creatine and Creatinine Metabolism - Physiological Reviews
Creatine and Creatinine Metabolism - Physiological Reviews
Creatine and Creatinine Metabolism - Physiological Reviews
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July 2000 CREATINE AND CREATININE METABOLISM 1125<br />
changes in kinetic properties were observed for the<br />
D129N, D129A, Y133F, <strong>and</strong> T135A mutant enzymes.<br />
Scrutiny of the reactivity of the amino groups of<br />
recombinant rat GAMT toward radioactive acetic anhydride<br />
revealed that Lys residues 38, 83, 104, 108, 152, <strong>and</strong><br />
180 are moderately reactive, that lysines 113 <strong>and</strong> 160 are<br />
weakly reactive, <strong>and</strong> that lysines 178 <strong>and</strong> 234 are nonreactive<br />
(972). The amino group of the NH 2-terminal Ser<br />
residue, on the other h<strong>and</strong>, displays high reactivity, indicating<br />
that it is exposed to the solvent. Although AdoMet<br />
has no influence on the reactivity of the Lys residues, a<br />
substantial reduction in radioactive labeling of Lys-38 is<br />
brought about when both AdoMet <strong>and</strong> guanidinoacetate<br />
are present. Because, however, even excess concentrations<br />
of these substrates do not suppress the reactivity of<br />
Lys-38 completely, this residue was suggested not to be<br />
involved directly in substrate binding.<br />
Finally, trypsin, chymotrypsin, <strong>and</strong> elastase all cleave<br />
native <strong>and</strong> recombinant rat liver GAMT preferentially between<br />
amino acids 19 <strong>and</strong> 25 (269, 713). Cleavage is<br />
paralleled by inactivation of the enzyme <strong>and</strong> by a large<br />
decrease in the affinities for both AdoMet <strong>and</strong> guanidinoacetate.<br />
The presence of either substrate has no effect on<br />
the rate of inactivation by trypsin, but a substantial protection<br />
from inactivation is observed when both substrates<br />
are present, or when Cys-15 is cross-linked with<br />
Cys-90 via a disulfide bond (269). These results suggest<br />
that residues 19–25 of GAMT are flexible <strong>and</strong> exposed to<br />
the solvent <strong>and</strong> that the NH 2-terminal region is not involved<br />
directly in substrate binding, but plays a role in<br />
catalysis.<br />
C. Cr Transporter<br />
Investigation of Cr transport across the plasma membrane<br />
has been hampered for many years by the problems<br />
inherent in the study of integral membrane proteins. However,<br />
recent cDNA <strong>and</strong> gene sequencing of the Cr transporters<br />
from rabbit (319), rat (295, 619, 840, 860), mouse<br />
(697), human (52, 214, 415, 691, 843, 927), <strong>and</strong> electric ray<br />
(Torpedo) (318) has given a fresh impetus in this particular<br />
field. These DNA sequencing approaches have shown<br />
that Cr transporters are composed of 611–636 amino acid<br />
residues <strong>and</strong> have a calculated M r of �70,000. The Cr<br />
transporters are members of the Na � -dependent “neurotransmitter”<br />
transporter family. They are most closely<br />
related to the GABA/taurine/betaine transporter subfamily<br />
(46–53% amino acid sequence identity), while the homology<br />
to Gly, Pro, catecholamine, <strong>and</strong> serotonin transporters<br />
is somewhat less pronounced (38–44%). All Cr<br />
transporters lack a hydrophobic NH 2-terminal signal sequence<br />
<strong>and</strong> display 12 putative transmembrane domains<br />
like other members of the Na � -dependent neurotransmitter<br />
transporter family. Consequently, the NH 2 <strong>and</strong> COOH<br />
termini of the polypeptide chain are probably both directed<br />
toward the cytosol.<br />
By classical biochemical means, a saturable uptake<br />
mechanism for Cr was identified in rat <strong>and</strong> mouse skeletal<br />
muscle (150, 250, 711), human fibroblasts, human uterine<br />
<strong>and</strong> calf aortic smooth muscle cells (150), human red<br />
blood cells (965), human monocytes <strong>and</strong> monocyte-derived<br />
macrophages (570), as well as in rat astroglia cells<br />
(659). In addition to the saturable component of Cr uptake,<br />
kinetic analysis often revealed a second component,<br />
displaying a K m for Cr of �1.3 mM or being not saturable<br />
(see Refs. 150, 570, 965). In the light of a Cr concentration<br />
in the serum of 25–50 �M (175), this latter component<br />
seems to be irrelevant for Cr uptake in vivo <strong>and</strong> may<br />
represent passive diffusion of Cr across the plasma membrane.<br />
Investigation of the tissue distribution of the Cr transporter<br />
by Northern blotting <strong>and</strong> in situ hybridization techniques<br />
gave somewhat contradictory results. It remains to<br />
be established whether the quantitative differences observed<br />
are due to methodological artifacts or to pronounced<br />
species differences. So far, expression of Cr<br />
transporter mRNA seems to be highest in kidney, heart,<br />
<strong>and</strong> skeletal muscle <strong>and</strong> somewhat lower in brain, small<br />
<strong>and</strong> large intestine, epididymis, testis, vas deferens, seminal<br />
vesicles, prostate, <strong>and</strong> adrenal. Only very low<br />
amounts or no Cr transporter mRNA at all are found in<br />
ovary, uterus, placenta, liver, lung, spleen, pancreas, <strong>and</strong><br />
thymus (295, 319, 415, 543, 619, 691, 840, 860, 927). In<br />
brain, the regional distribution of Cr transporter transcripts<br />
is heterogeneous, but again, the quantitative measures<br />
published on this issue differ to some extent (295,<br />
332, 359, 619, 840, 860). Nevertheless, in situ hybridization<br />
experiments suggested that Cr transporter expression in<br />
rat brain closely correlates with the localization of CK<br />
(332). In the digestive tract of the rat, moderate levels of<br />
Cr transporter transcripts were observed in the small <strong>and</strong><br />
large intestine, but only low levels were seen in the stomach<br />
(295). In contrast, Cr transporter mRNA was undetectable<br />
in human gut. In rat <strong>and</strong> human kidney, expression<br />
of the Cr transporter seems to be slightly higher in<br />
the cortex than in the medulla (295, 691). Finally, the level<br />
of Cr transporter expression in transformed cells is likely<br />
to depend on the tumor type. In human colonic tumor cell<br />
lines, only very low levels of Cr transporter mRNA were<br />
detected (295). On the other h<strong>and</strong>, 1 h after intravenous<br />
injection of radioactive Cr into tumor-bearing mice, Ehrlich<br />
ascites tumor cells <strong>and</strong> intestine displayed the highest<br />
specific radioactivities, suggesting that they have considerable<br />
Cr transporter activities (1135).<br />
Experiments in the late 1960s on Cr uptake into rat<br />
skeletal muscle yielded an unusually high K m(Cr) of 0.5<br />
mM (250, 251). More recent studies, with improved techniques,<br />
have shown that the cloned <strong>and</strong> overexpressed Cr<br />
transporters from rabbit, rat, human, <strong>and</strong> Torpedo as well