N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 61

Table 5.3 Plasma N-Hcy-protein, Hcy-thiolactone, and tHcy are reversibly modified by a diet in
wild-type C57BL/6 J mice (Data from [113])
Diet (eight mice/group) N-Hcy-protein ( μ M) Hcy-thiolactone (nM) tHcy ( μ M)

Control, 0 weeks 1.89 0.70 5.74 2.42 3.45 0.30

High-Met, 2 weeks 22.1 13.5 79.4 11.9 126 76

High-Met, 2 weeks control, 4 weeks 3.60 1.11 4.47 0.97 5.47 2.83

only a normal chow diet. At the same time plasma tHcy and Hcy-thiolactone
return to normal values. These findings are consistent with much slower turnover
of N-Hcy-protein (half-life 10.2 h) compared with the turnover of tHcy (half-life
26.2 min) and Hcy-thiolactone (half-life 5.0 min) in the mouse (Fig. 5.4 )
[140, 141].
The discovery that PON1 has the ability to hydrolyze Hcy-thiolactone led to a
hypothesis that PON1 protects against protein N-homocysteinylation [81]. Sup-
port for this hypothesis came from findings showing that when serum is
supplemented with Hcy-thiolactone, much less N-Hcy-protein accumulates in
the serum from human donors with high Hcy-thiolactonase/PON1 activity com-
pared with donors with low Hcy-thiolactonase/PON1 activity [152]. The lowest
extent of protein N-homocysteinylation is observed in rabbit serum [152] which
has the highest Hcy-thiolactonase/PON1 activity (six times that of an average
human serum) [153]. Thus, high Hcy-thiolactonase activity affords better protec-
tion against protein N-homocysteinylation in vitro than the low activity. This
inverse relationship between the levels of N-Hcy-protein and Hcy-thiolactonase
activity of PON1 is also observed in vivo in humans. In fact, plasma N-Hcy-
protein

is negatively correlated with serum Hcy-thiolactonase activity

(r ¼ 0.01) in CBS-deficient patients [250]. However, in contrast to

the activity of PON1 measured with the natural substrate Hcy-thiolactone,
enzymatic activities of the PON1 protein measured with artificial substrates
correlate less strongly (r ¼

0.43, P ¼

0.025 for paraoxonase activity) or do
not correlate at all (phenyl acetate hydrolase and

0.36, P ¼

-thiobutyrolactone hydrolase
activities) with plasma N-Hcy-protein. Furthermore, the inverse in vivo relation-
ship between Hcy-thiolactonase activity and N-Hcy-protein is recapitulated in
separate in vitro N-homocysteinylation experiments with [ 35 S]Hcy-thiolactone
and sera from CBS-deficient subjects. Taken together, these findings provide
evidence that the Hcy-thiolactonase activity of PON1 is a major determinant of
plasma N-Hcy-protein levels in vivo in humans [250].
In related experiments, the level of N-linked Hcy in plasma proteins has been
found to increase in rats in response to lowering serum Hcy-thiolactonase activity
of PON1 by treatment with the lipid metabolism-regulating hormone leptin. As
leptin administration has no effect on plasma total Hcy, it has been suggested that
the decreased capacity to metabolize Hcy-thiolactone and concomitant increase in
protein N-homocysteinylation contribute to the pro-atherogenic effect of chronic
hyperleptinemia [302].

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