Studies of the aminopeptidase proteolysis of semax analogues with different N-terminal amino acid residues

Article abstract of DOI:10.1134/S1068162011040133

Proteolysis of semax (Met-Glu-His-Phe-Pro-Gly-Pro, Sem) and its analogues with the substitution of Ala, Gly, Thr, or Trp for the N-terminal Met was studied. This substitution was shown to change the degradation rate of these peptides by leucine aminopeptidase (EC 3.4.11.2, Sigma, Type VI, 9.2 activity units/mg). [Ala¹]Sem, [Gly¹]Sem, and [Thr¹]Sem (the semax analogues) proved to be more stable to the proteolysis than semax itself. It was demonstrated that the primary product of the proteolysis was His-Phe-Pro-Gly-Pro (Sem-5). In the case of [Trp¹]Sem, the comparable amount of Glu-His-Phe-Pro-Gly-Pro (Sem-6) was found to be formed along with Sem-5. In was established that all the studied semax analogues could be used as inhibitors of its proteolysis. Pleiades Publishing, Ltd., 2011.

Full text of DOI:10.1134/S1068162011040133

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 4, pp. 421–427. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © K.V. Shevchenko, T.V. V’yunova, I.Yu. Nagaev, L.A. Andreeva, L.Yu. Alfeeva, N.F. Myasoedov, 2011, published in Bioorganicheskaya Khimiya, 2011,
Vol. 37, No. 4, pp. 475–482.
Studies of the Aminopeptidase Proteolysis of Semax Analogues
with Different NꢀTerminal Amino Acid Residues
K. V. Shevchenko1, T. V. V’yunova, I. Yu. Nagaev, L. A. Andreeva,
L. Yu. Alfeeva, and N. F. Myasoedov
Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, Moscow, 123182 Russia
Received August 31, 2010; in final form, October 22, 2010
Abstract—Proteolysis of semax (MetꢀGluꢀHisꢀPheꢀProꢀGlyꢀPro, Sem) and its analogues with the substituꢀ
tion of Ala, Gly, Thr, or Trp for the Nꢀterminal Met was studied. This substitution was shown to change the
degradation rate of these peptides by leucine aminopeptidase (EC 3.4.11.2, Sigma, Type VI, 9.2 activity
units/mg). [Ala1]Sem, [Gly1]Sem, and [Thr1]Sem (the semax analogues) proved to be more stable to the proꢀ
teolysis than semax itself. It was demonstrated that the primary product of the proteolysis was HisꢀPheꢀProꢀ
GlyꢀPro (Semꢀ5). In the case of [Trp1]Sem, the comparable amount of GluꢀHisꢀPheꢀProꢀGlyꢀPro (Semꢀ6)
was found to be formed along with Semꢀ5. In was established that all the studied semax analogues could be
used as inhibitors of its proteolysis.
Keywords: semax, aminopeptidase, rat brain membranes, semax analogues, peptide degradation
DOI: 10.1134/S1068162011040133
1
INTRODUCTION
treated rats. At the same time, the rest of the Sem anaꢀ
logues ([Gly1]Sem, [Ala1]Sem, and [Thr1]Sem)
caused no significant changes in rat behavior in comꢀ
parison with the control group of rats. NFR for these
peptides was significantly lower than that for the group
that was treated with semax. Thus, the replacement of
Met1 by Ala, Gly, Thr, or Trp in semax limited specꢀ
trum of the nootropic action or resulted in a complete
loss of the nootropic effect of the peptides. The
authors of paper [3] proposed that modification of the
Biodegradation of semax in vivo is known to result
in the formation of a number of its shortened anaꢀ
logues that are often no less biologically active than the
starting peptide [1]. It has been found that the main
products of the Sem degradations in the rat brain are
peptides that are formed by the cleavage by aminopepꢀ
tidases. The number of Sem metabolites in the brain is
continuously increased with time [1]. Moreover, it was
reported [2] that sites of specific binding of some of
these peptides (Semꢀ5 and Semꢀ3) are present in rat
brain membranes. These peptides were found to be
specifically bond in different regions of the rat brain
[2]. Semax and Semꢀ5 were found on the membranes
of hippocampus and cerebellum, whereas Semꢀ3 was
localized on membranes of basal nuclei. Therefore,
semax is a source of unique regulatory complex of the
peptides.
Nꢀterminal amino acid residue in the Sem molecule
changed not only the rate but the pathway of proꢀ
teolytic degradation of the starting peptide as well. As
a result, these peptides formed a different set of metaꢀ
bolic products which had no neurotropic activity.
Thus, kinetic studies of degradation of semax and its
analogues by different aminopeptidases in vitro are
important for understanding of cerebral processes
in vivo.
Neurotropic activity of a number of the Sem anaꢀ
logues with the substitution of Gly, Ala, Thr, or Trp for
Met was studied and their neurotropic effects were
compared with those of semax in the paper [3]. The
influence of [Trp1]Sem on NFR in an experiment of
rat learning with the negative reinforcement was found
to be the same as that in the control group of the Semꢀ
The goal of this study was investigation of the degꢀ
radation of semax and its analogues in the presence of
aminopeptidase M (its former name was leucine amiꢀ
nopeptidase, EC 3.4.11.2, Type VI) and under the
action of membrane enzymes of the rat brain in vitro.
RESULTS AND DISCUSSION
Abbreviations: AU, activity unit; MERB, membrane enzymes of
rat brain; NFR, the number of fulfilled reactions; Sem, semax;
Semꢀ6, GluꢀHisꢀPheꢀProꢀGlyꢀPro; Semꢀ5, HisꢀPheꢀProꢀGlyꢀ
Pro; Semꢀ4, PheꢀProꢀGlyꢀPro; Semꢀ3, ProꢀGlyꢀPro; PBS,
phosphateꢀsalt buffer.
Corresponding author: fax: (499) 196ꢀ0216; eꢀmail: ATRegisꢀ
ter@mail.ru.
The microsomal aminopeptidase M (EC 3.4.11.2,
Leucine Aminopeptidase Microsomal, Type VI;
Sigma, United States) was used as an aminopeptidase
for the studies of proteolysis of semax and its anaꢀ
logues. Experiments were performed in a phosphateꢀ
1
421

422
SHEVCHENKO et al.
(a)
(b)
100
90
80
70
60
50
40
30
20
10
100
2
90
80
70
60
50
40
30
2
1
1
20
10
0
10
20
30
Time, min
40
50
60
0
10
20
30
Time, min
40
50
60
Fig. 1. Kinetics of semax degradation (a): (
1
), Sem and (2), Semꢀ5; (b): (1), Semꢀ5 and (2), Semꢀ4 by aminopeptidase M.
salt buffer (PBS, pH 7.4). The proteolytic conditions culated from the Michaelis–Menten equation on the
were chosen during the Sem degradation at different basis of the obtained data.
ratios of the substrate and the enzyme. The optimum
ratio for these studies proved to be 10.3 mol of the
peptide per 1 AU of the peptidase. Under these condiꢀ
tions, semax was cleaved by more than 90% within
60 min.
Analysis of the composition of proteolytic metaboꢀ
lites demonstrated that semax was cleaved to only
Semꢀ5 and the Nꢀterminal MetꢀGlu dipeptide at all of
μ
the used substrate–enzyme ratios (Fig. 1a).
The kinetics of degradation of Semꢀ5 by amiꢀ
nopeptidase M was studied in order to determine posꢀ
sibility of cleavage of Semꢀ5 by this enzyme (Fig. 1b).
As follows from Fig. 1b, Semꢀ5 is rapidly cleaved in the
absence of semax, but the rate of conversion of semax
into Semꢀ5 is approximately 2.4 times higher than that
of Semꢀ5 into Semꢀ4 (Fig. 2). Therefore, proteolysis
of the pentapeptide is probably impossible if both
Semꢀ7 and Semꢀ5 are present in the reaction mixture.
This fact is explained by two reasons: competition of
Semꢀ7 and Semꢀ5 for active sites of the enzyme and
the higher rate of conversion of Semꢀ7 into Semꢀ5 in
comparison with that of Semꢀ5 into Semꢀ4.
The rate of the Sem cleavage by aminopeptidase M
was analyzed in the Lineweaver–Burk coordinates at
different concentrations of the substrate. The maxiꢀ
mum rate of the Sem proteolysis (
and the Michaelis constant (Кm = 200
V
max = 58 M/min)
μ
μ
M) were calꢀ
900
750
600
450
300
150
1
2
The similar experiments with Semꢀ4 demonstrated
that there is no further degradation of Semꢀ4 in the
presence of this enzyme.
In order to investigate the influence of the MetꢀGlu
dipeptide and free amino acids (methionine, alanine,
glycine, threonine, and tryptophan), the dipeptide or
one of the aforementioned amino acids were added to
the reaction mixture in the molar ratios of 1 : 1, 10 : 1,
and 100 : 1 relative to the semax quantity. All the above
substances were found to have no effect on the semax
degradation.
Kinetics of the degradation of [Ala1]Sem,
[Gly1]Sem, [Thr1]Sem, and [Trp1]Sem by aminopepꢀ
tidase M was studied for evaluation of the effect of
0
10
20
30
40
50
60
Time, min
Fig. 2. Kinetics of the formation of (
and ( ) Semꢀ4 from Semꢀ5 by the action of aminopeptiꢀ
dase M.
1) Semꢀ5 from semax
modification of the
Nꢀterminal amino acid residue of
2
semax on proteolysis of these peptides. The proꢀ
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STUDIES OF THE AMINOPEPTIDASE PROTEOLYSIS OF SEMAX ANALOGUES
423
100
90
80
70
60
50
40
30
20
10
100
3
90
2
80
70
1
60
50
40
1
30
2
3
4
5
20
10
0
20
40
60
80
100
120
0
10
20
30
40
50
60
Time, min
Time, min
1
Fig. 3. Kinetics of the degradation of (1) [Ala ]Sem,
1
1
1
1
(2
) [Gly ]Sem,
(3)
[Thr ]Sem,
(4)
[Trp ]Sem, and
Fig. 4. Kinetics of the degradation of [Trp ]Sem by amiꢀ
1
(5) semax by aminopeptidase M.
nopeptidase M; (1) [Trp ]Sem, (2) Semꢀ6, and (3) Semꢀ5.
teolytic reaction was performed under the same conꢀ analogues. This observation is particularly evident after
ditions as those of the semax proteolysis (Fig. 3).
prolonged incubation with amino peptidase M. Incubaꢀ
tion of semax for 60 min in the presence of its analogues
([Ala1]Sem, [Gly1]Sem, [Thr1]Sem, and [Trp1]Sem) at
the Sem/analogue ratio of 1 : 5 resulted in the 6, 6.4,
4.8, and 7.5 increase in the semax stability, respectively.
As one can see from Fig. 3, [Ala1]Sem, [Gly1]Sem,
[Thr1]Sem, and [Trp1]Sem are less effectively cleaved
by aminopeptidase M than semax itself under the
same conditions. Thus, our data confirm the proposal
[3] that the change in biological activity of the Sem
analogues after modification of its Nꢀterminal part can
Table 1. Influence of the semax analogues on the proteolyꢀ
be associated with the change in the rate of proteolysis
of the modified peptides. Therefore, we can propose
several explanations of preservation of the nootropic
activity of [Trp1]Sem. First, the rate of its proteolysis is
higher than that of the rest of the Sem analogues, and
the completeness of its hydrolysis within 60 min differs
by less than 10% from that of semax. Second, a comꢀ
parable amount of Semꢀ6, which is known to exhibit
high nootropic activity [4], is formed along with
Semꢀ5 in the course of proteolysis of [Trp1]Sem disꢀ
tinct from the other semax analogues. Semꢀ5 and
Semꢀ6 arise in the ratio of 1 : 1 5 min after the beginꢀ
ning of incubation of [Trp1]Sem (see Fig. 4). The conꢀ
tent of Semꢀ6 in the reaction mixture proved to be
more than 25% in 120 min.
sis of semax by leucime aminopeptidase
Reaction time, min
Added Sem/analogue,
analogue*
mol/mol
15
30
60
120
–
–
69.80 54.50 12.60 3.50
1
[Ala ]Sem
1 : 0.5
1 : 3
55.93 48.37 29.40 1.70
53.30
75.72
1 : 5
1
[Gly ]Sem
1 : 0.5
1 : 3
89.60 68.81 31.50 2.80
57.58
80.34
1 : 5
1
[Thr ]Sem
1 : 0.5
1 : 3
83.21 75.00 25.90 1.00
The low biological activity of the aforementioned
semax analogues and their stability to aminopeptidase
M (EC 3.4.11.2) give an idea of using these peptides as
inhibitors of the semax proteolysis. This assumption
was checked in the studies of the semax stability
towards aminopeptidase M in the presence of its anaꢀ
logues in the incubation medium (Table 1).
55.12
59.88
1 : 5
1
[Trp ]Sem
1 : 0.5
1 : 3
86.6 82.0 71.2 58.4
96.0 93.6 88.4 74.3
98.9 97.9 94.6 85.7
1 : 5
* The reaction conditions were: 10.3
aminopeptidase M, 30 C. The residual content of semax in the
incubation mixture was given in % of the starting amount.
µmol of Sem per one AU of
As one can see from Table 1, the semax stability to
aminopeptidase M increases in the presence of the Sem
°
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424
SHEVCHENKO et al.
Table 2. Content of Semꢀ6 in the reaction mixture of proꢀ
teolysis of [Thr1]Sem by aminopeptidase M* depending on
the content of the added semax
conclude on the basis of these results that affinity of
semax to the active site of the enzyme is much higher
than that of [Trp1]Sem.
Proteolysis of semax and its analogues was studied
in the presence of MERB in vitro. We choose the
MERB concentration (0.195 mol of semax per 1 mg
Reaction time, min
Sem:[Thr1]Sem,
mol/mol
15
30
60
90
120
μ
of the protein) so that semax in the analogous concenꢀ
trations was cleaved within 60 min approximately in
the same degree as with the use of aminopeptidase M.
0 : 1
1 : 0.5
1 : 1
1 : 3
1 : 5
15.8
0.0
0.0
0.0
3.9
30.3
0.0
2.6
7.9
9.2
53.9
5.3
71.1 100.0
5.3
11.8
22.4
23.7
9.2
17.1
31.6
36.8
Interaction of semax with MERB for 15 min
resulted in its 45% degradation distinct from the in
vivo experiments [5] in which the Sem concentration
in the rat brain decreased fivefold during the same time
period (Fig. 6). Thus, the Sem proteolysis in the presꢀ
ence of MERB was more slow in comparison with that
in vivo, but faster in comparison with its cleavage by
aminopeptidase M (Fig. 3).
7.9
15.6
15.8
1
* The reaction conditions were: 10.3
of aminopeptidase M, 30 C. The content of Semꢀ6 in the incubaꢀ
tion mixture was given in percent of its amount that was formed
under the analogous conditions without the addition of semax.
µ
mol of [Trp ]Sem per one AU
°
Semꢀ5 is mainly generated during the interaction
of semax with MERB, and total concentration of
other products of the degradation begins to predomiꢀ
nate over the Semꢀ5 concentration only after one hour
(Fig. 6). The main part of semax was found to convert
into Semꢀ5 within approximately 40 min, whereas
concentrations of Semꢀ3 and Semꢀ4, at that time,
proved to be about 11 and 4%, respectively. After the
next 20 min, the content of Semꢀ5 decreased to 43%,
and that of Semꢀ3 and Semꢀ4 achieved 32 and 18%,
respectively; i.e., the rate of Semꢀ5 degradation
increased.
Hence, the maximum effect is observed, as expected, in
the case of [Trp1]Sem and decreased in the row
[Gly1]Sem > [Ala1]Sem > [Thr1]Sem. It is interesting
that the inhibition of the Sem proteolysis by [Gly1]Sem,
[Ala1]Sem, and [Thr1]Sem at the ratio of 1 : 0.5 is comꢀ
pletely disappeared after 120ꢀmin incubation. At the
same time, the inhibiting effect of [Trp1]Sem on the
Sem proteolysis is preserved after 120 min (the semax
stability is increased by more than one order).
We can investigate the change in stability of
[Trp1]Sem in the presence of semax, because Semꢀ6 is
formed only from [Trp1]Sem during the incubation of
the mixture of semax and [Trp1]Sem (Table 2, Fig. 5).
We proposed the following explanation for this
effect. The shorter peptides are evidently formed from
Semꢀ5 under the action of the MERB enzymes, simiꢀ
As follows from our data, the formation of Semꢀ6 larly to the reaction with aminopeptidase M. MERB is
decreased by 2.7 times in comparison with the experiꢀ a complex aggregate of membraneꢀbound enzymes.
ment without semax, even if the [Trp1]Sem/Sem ratio Semꢀ5 and semax can differ in their influence on its
was as high as 5 : 1. If the semax content in the incubaꢀ functioning. Semꢀ5 that is formed during the incubaꢀ
tion medium is two times higher, the formation of tion reacts with MERB and somehow corrects funcꢀ
Semꢀ6 decreases by approximately elevenfold. We tioning of this enzymatic complex. As a result, the proꢀ
4
(a)
(b)
(c)
(d)
4
4
3
4
3
3
3
2
2
2
2
1
1
1
1
5
6
7
8
9
5
6
7
8
9
5
6
7
8
9
5
6
7
8
9
1
Fig. 5. HPLC of the reaction mixture of proteolysis of the mixtures of Sem and [Trp ]Sem in the following ratios: (a) 1 : 0.5,
(b) 1 : 1, (c) 1 : 3, (d) 1 : 5, by aminopeptidase M 60 min after the start of the reaction. Peaks
Semꢀ5, Sem, and [Trp ]Sem, respectively.
1
,
2,
3, and
4
corresponded to Semꢀ6,
1
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STUDIES OF THE AMINOPEPTIDASE PROTEOLYSIS OF SEMAX ANALOGUES
425
100
80
60
40
20
100
3
1
4
80
60
4
2
40
20
5
1
2
0
10
20
30
Time, min
40
50
60
3
Fig. 7. Kinetics of proteolysis of (1) semax and its anaꢀ
5
1
1
1
logues: (
2
) [Ala ]Sem, (
3
) [Gly ]Sem, (
4) [Thr ]Sem, and
1
(
5) [Trp ]Sem in the presence of MERB.
0
10
20
30
40
50
60
Time, min
(3) The pronounced kinetic effects were observed
in the case of [Gly1]Sem and [Thr1]Sem. The curve of
time dependence of the peptide content at the beginꢀ
ning of the reaction (the first 20–30 min) looked like
that for the proteolysis of these peptides by aminopepꢀ
tidase M. Then, the rate of proteolysis of these pepꢀ
tides by MERB considerably increased.
(4) The similar time dependence of the proteolysis
rate could also be observed for the other aforemenꢀ
tioned peptides, but the second stage of the process
was ensured several minutes after the beginning of the
reaction, and the picture was indistinct.
Fig. 6. Kinetics of (
of MERB and accumulation of the following products of
this reaction: ( ) Semꢀ3, ( ) Semꢀ4, ( ) Semꢀ5, and
) Semꢀ6.
1) the Sem degradation in the presence
2
3
4
(5
teolysis of Semꢀ5 is accelerated, and we can propose
that the substrate nature strongly affects functioning of
the MERB enzymatic complex.
Thus, the main product of the semax proteolysis by
both aminopeptidase M and MERB is Semꢀ5. Howꢀ
ever, MERB is a more complex enzymatic aggregate,
and its enzymatic activity can be significantly changed
with variations in the composition of the incubation
medium. If this proposal is true, the semax analogues
will differently interact with MERB, and the kinetics
of proteolysis of each analogue will be distinguished its analogues demonstrated that the
from that of proteolysis by the aminopeptidase M in a
different degree. In other words, the similar effects will
possibly be observed as a result of changes in the conꢀ
tent of [Gly1]Sem, [Ala1]Sem, [Trp1]Sem, and
[Thr1]Sem in the course of the incubation with
MERB, as it took place during the formation of Semꢀ5
from semax under the analogous conditions (Fig. 2b).
Thus, these data confirm our proposal that the subꢀ
strate nature strongly affects functioning of the MERB
enzymatic complex.
Analysis of the products of proteolysis of semax and
Nꢀterminal proteolꢀ
ysis is characteristic for most of the peptides (Fig. 8).
The beginning of the proteolysis for the majority of
the Sem analogues is determined by the activity of
diaminopeptidases (and monoaminopeptidases in the
case of [Trp1]Sem). The shorter peptides are probably
formed from Semꢀ5 by the action of diaminopeptiꢀ
dases (Semꢀ3) or by the histidine cleavage (Semꢀ4). In
distinction of the in vivo results [1], the amount of
Semꢀ3 is always significantly lower than that of Semꢀ4
when the peptides interact with MERB. In some cases
([Gly1]Sem and [Thr1]Sem), no Semꢀ3 is even
formed.
The experiments demonstrated that:
(1) The proteolysis rate of [Gly1]Sem, [Ala1]Sem,
[Trp1]Sem, and [Thr1]Sem by MERB proved to be
higher than that by the leucine aminopeptidase M, as
in the case of semax itself (Fig. 3, 7).
(2) [Gly1]Sem and [Thr1]Sem were most slowly
Therefore, [Gly1]Sem, [Ala1]Sem, [Trp1]Sem, and
cleaved by both MERB and aminopeptidase M, [Thr1]Sem were shown to be more stable to the proꢀ
whereas [Ala1]Sem was hydrolyzed more effectively, teolysis by aminopeptidases in comparison with semax
and proteolysis of [Trp1]Sem was comparable with that itself. Substitution for the
Nꢀterminal residue of semax
of semax.
could influence the rate of the peptide degradation by
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426
SHEVCHENKO et al.
5 to 100%) in the buffer (0.2 M LiClO4 + 0.005 M
HClO4, pH 2.24) within 12 min at 35 . The peptides
were detected at 210 nm.
Semax, [Gly1]Sem, [Ala1]Sem, [Trp1]Sem, and
[Thr1]Sem were synthesized by the method described
in [7].
The prepared compounds were analyzed by TLC
on silica gel plates (Silufol, Czech Republic) and
detected by spraying the plates by the ninhydrin soluꢀ
tion. Chromatographic mobility (Rf) was determined
in the following chromatographic systems:
(A) butanol–acetic acid–water (4 : 1 : 1), (B) butanol–
acetic acid–pyridine–water (30 : 6 : 20 : 24), (C) chloroꢀ
form–methanol–ammonium hydrate (6 : 4 : 1),
12
10
8
°C
Semꢀ6
Semꢀ5
Semꢀ4
Semꢀ3
6
4
(D) chloroform–methanol–ammonium
hydrate
(4 : 4.5 : 1.5), and (E) butanol–acetic acid–pyridine–
water (30 : 6 : 20 : 24).
2
Characteristics of the prepared peptides:
HCl
848.5; mp 115–117°С (decomposition); [α]D
65.5 1, CH3OH), Rf 0.08 (A), 0.42 (B), 0.38 (C);
HCl AlaꢀGluꢀHisꢀPheꢀProꢀGlyꢀProꢀOH: 788.5
mp113–115°С, [ , methanol); f 0.234
20 – 64.55°(
(C), 0.484 (D), 0.1D62 (E);
HCl GlyꢀGluꢀHisꢀPheꢀProꢀGlyꢀProꢀOH:
mp 110–111
Rf 0.172 (C), 0.438 (D); 0.163 (E);
HCl ThrꢀGluꢀHisꢀPheꢀProꢀGlyꢀProꢀOH:
mp 121–123
Rf 0.313 (C), 0.860 (D), 0.175 (E);
HCl TrpꢀGluꢀHisꢀPheꢀProꢀGlyꢀProꢀOH:
mp 119–122°С, [α]D –61.45
Rf 0.305 (C), 0.785 (D), 0165 (E).
⋅
MetꢀGluꢀHisꢀPheꢀProꢀGlyꢀProꢀOH:
0
22
[Gly1]Sem
[Ala1]Sem
[Trp1]Sem
Sem
М
⎯
[Thr1]Sem
°
(с
⋅
М
R
;
Fig. 8. The content of the products of Nꢀterminal proteolꢀ
α
]
с
1
ysis (for 10 min) of semax and its analogues that were
formed in the reaction mixture in the presence of MERB.
The values were given in % of the total amount of the comꢀ
pounds.
⋅
М
774.5;
20
°
С
;
[α]D –69.66
°
(с
1,
methanol);
aminopeptidases. Moreover, the rate of semax proꢀ
teolysis was shown to decrease after the addition of its
analogues to the incubation mixture; i.e., the competꢀ
itive inhibition of the Sem proteolysis by Sem anaꢀ
logues was observed.
⋅
М
818.5
;
20
°
С, [α]D –62.28 (с 1, methanol),
°
⋅
М
903.5
;
20
Different peptides were shown to differently affect
the activity of the MERB enzymatic complex. The difꢀ
ferent activity of the enzymatic complex of cellular
membranes in vivo during its interaction with semax
or its analogues could probably be one of the reasons
for the decrease in the spectrum of nootropic action
(up to its complete loss) of the semax analogues with
°
(с 1, methanol),
Preparation of the membrane fraction of the rat
brain. All the operations of the membrane isolation
were performed at
4°С. Adult male rats of the Wistar
line were decapitated. Their forebrain was isolated,
washed with cold phosphateꢀsalt buffer (10 mM
sodium phosphate and 150 mM sodium chloride,
pH 7.4), and homogenized in 10 mM TrisꢀHCl buffer
(pH 7.4, 10 volumes) containing 0.32 M saccharose,
1 mM EDTA (buffer A), 1 mM benzamidine, and
0.1 mM phenylmethylsulfonyl fluoride (PMSF) in a
Teflonꢀglass homogenizer. The homogenate was cenꢀ
modified
Nꢀterminal amino acid residue [3].
EXPERIMENTAL
Aminopeptidase M (its previous name was leucine
aminopeptidase, EC 3.4.11.2, Sigma, Type VI,
microsomal, from pig kidney, 9.2 AU/mg) and necesꢀ
sary commercial products were used in this study.
MERB (8.6 mg of the protein/ml) were obtained from
the whole brain of the Wistar rats (males with the body
weight of 200 g). HꢀGlu(OBut)ꢀHisꢀPheꢀProꢀGlyꢀ
ProꢀOH was synthesized in the Institute of Molecular
Genetics of the Russian Academy of Sciences [6].
trifuged for 20 min at 1000
removed, and the supernatant was repeatedly centriꢀ
fuged for 30 min at 24000 . The dense mitochondriaꢀ
g. The precipitate was
g
rich brown precipitate on a bottom of the tube was disꢀ
carded, and the less dense light precipitate of memꢀ
branes was suspended in buffer A, replaced in a clean
tube, and washed with the same buffer two times with
The reaction mixtures were analyzed by HPLC on the subsequent centrifugation (as described above) for
a Milikhrom Aꢀ02 chromatograph equipped with a precipitation of the membranes. The precipitate was
ProntoSILꢀ120ꢀ5ꢀC18 AQ DBꢀ2003 column (
2
×
75 mm, suspended in 10 mM TrsꢀHCl buffer (pH 7.4) conꢀ
the particle size of 5 μm) in a methanol gradient (from taining 0.22 M saccharose and stored in liquid nitroꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 4
2011

STUDIES OF THE AMINOPEPTIDASE PROTEOLYSIS OF SEMAX ANALOGUES
427
gen [8]. The protein concentration in the membrane Studies of Presidium of the Russian Academy of Sciꢀ
samples was determined by the Lowry method [9]; it ences, by the project “Scientific Schools,” no. NShꢀ
proved to be 8.6 mg/ml.
3626.2008.4, and by the project of the state support of
young Russian scientists of the President of Russian
Federation, no. MKꢀ2375.2009.4 (under the direction
of Academician N.F. Myasoedov).
Proteolysis by aminopeptidase M and MERB. The
proteolytic conditions of cleavage by aminopeptidase M
were found in the experiments with degradation of semax
at different ratios of the substrate and the enzyme. Amiꢀ
nopeptidase M (30
μl, 0.30
μ
g, 1.942
10–6 AU) in the
×
REFERENCES
phosphateꢀsalt buffer (27.4 mM NaCl, 0.4 mM KCl,
2 mM Na3PO4 in 100 ml of water, pH 7.4) was added
1. Shevchenko, K.V., Nagaev, I.Yu., Alfeeva, L.Yu.,
Andreeva, L.A., Kamenskii, A.A., Levitskaya, N.G.,
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to the solution of the corresponding peptide (150
0.18 mol) in the same buffer (290 l). The amount of
semax was varied from 2.6 to 82 mol per one unit of
μ
g,
μ
μ
μ
the enzyme, and the reaction was continued up to
120 min. Semax degradation of more than 90% was
achieved at the ratio of semax and amino peptidase M
2. V’yunova, T.V., Shevchenko, K.V., Shevchenko, V.P.,
Bezuglov, V.V., and Myasoedov, N.F., Radiokhimiya
,
of about 10 : 1 (μmol/AU) for 60 min.
2009, vol. 51, pp. 161–166. [Radiochemistry (Engl.
Transl.), vol. 51, pp. 183–189].
Degradation of the aforementioned semax anaꢀ
logues (Fig. 3) and the semax stability depending on
the content of these analogues in the reaction mixture
(Table 1) were studied under the conditions that were
found for semax.
3. Glazova, N.Yu., Sebentsova, E.A., Levitskaya, N.G.,
Andreeva, L.A., Alfeeva, L.Yu., Kamenskii, A.A., and
Myasoedov, N.F., Izv. Akad. Nauk, Ser. Biol., 2005,
no. 4, pp. 460–466.
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Kamenskii, A.A., and Myasoedov, N.F., Dokl. Akad.
Nauk, 1999, vol. 367, pp. 137–140.
In the case of the experiments with MERB, the
solution of MERB (107
was added to the same solution. The incubation mixꢀ
ture was stirred at a temperature of 30 , and aliquots
(20 l) were taken in 0.5, 2, 5, 10, 20, 30, 40, and
μ
l, 8.6 mg of the protein/ml)
°
С
5. Shevchenko, K.V., Synthesis of Tritium Labeled Steroid
μ
Hormones and Peptides to Determine Their Content, Disꢀ
tribution and Metabolism in Biological Objects in vivo
,
60 min. The proteolysis was stopped by the addition of
the equal volume of methanol to the samples. After the
proteolysis of the peptides in the presence of MERB,
the reaction mixture was purified by the solidꢀphase
extraction on the reversed phase. The peptide mixture
was applied onto a cartridge with a reversed phase,
eluted with the mixture of methanol and 0.1% aqueous
Cand. Sci. (Chem.) Dissertation, Moscow: MITKhT,
2007.
6. Shevchenko, V.P., Nagaev, I.Yu., Alfeeva, L.Yu.,
Andreeva, L.A., Klimova, P.A., Malkin, A.V.,
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K.V.,
and
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N.F.,
Radiokhimiya, 2006, vol. 48, pp. 260–266. [Radioꢀ
chemistry (Engl. Transl.), vol. 48, pp. 288–295].
TFA (4 : 1), evaporated, and dissolved in the 200
methanol–water mixture (10 : 90).
μl of
7. RF Patent No. 2206573, Byull. Izobret.,
No. 2001135362 (2001).
The samples which were obtained with the use of
aminopeptidase M and MERB were analyzed by
HPLC.
8. Dolotov, O.V., Zolotarev, Yu.A., Dorokhova, E.M.,
Andreeva, L.A., Alfeeva, L.Yu., Grivennikov, I.A., and
Myasoedov, N.F., Bioorg. Khim., 2004, vol. 30,
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2004, vol. 30, pp. 213–217].
ACKNOWLEDGMENTS
This study was partially supported by the Program
“Molecular and Cellular Biology” for Fundamental
9. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Ranꢀ
dall, R.J., J. Biol. Chem., 1951, vol. 193, pp. 265–275.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 4
2011