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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 3, 2007
Contents
Influence of Conformationally Constrained Amino
Acids Replacing Positions 2 and 3 of Arginine Vasopressin
(AVP) and Its Analogues on Their Pharmacological Properties
Pp. 213-217
D. Sobolewski, A. Prahl, I. Derdowska, A. Kwiatkowska,
J. Slaninová and B. Lammek
[Abstract]
Oligomerisation of Sarcoplasmic Reticulum Ca2+-ATPase
Monomers from Skeletal Muscle Pp. 219-226
D. Schreiber and K. Ohlendieck
[Abstract]
The Ways of Realization of High Specificity and Efficiency
of Enteropeptidase Pp. 227-232
A.G. Mikhailova, V.V. Likhareva, N. Teich and L.D. Rumsh
[Abstract]
Stem Bromelain: An Enzyme That Naturally Facilitates
Oriented Immobilization Pp. 233-236
H. Khatoon, H. Younus and M. Saleemuddin
[Abstract]
Critical Enzymes Involved in Endocannabinoid Metabolism
Pp. 237-246
B.S. Basavarajappa
[Abstract]
High-Level Expression and Purification of an Analgesic
Peptide from Buthus martensii Karch Pp.
247-251
Z. Cao, W. Wang, X. Xiao, K. Chen, X. Liang and D. Yu
[Abstract]
Structural Insights into the Exchange Domain of Sec2p:
Expression, Purification, Crystallization, and Preliminary
X-Ray Diffraction Data Analysis Pp. 253-258
H.S. Gill
[Abstract]
Design, Synthesis and Utilization of 1- Substituted
Sulphonyloxy- 2-Phenyl Benzimidazole as a Novel Peptide Coupling
Reagents Pp. 259-263
N.D. Kokare, R.R. Nagawade, V.P. Rane and D.B. Shinde
[Abstract]
Cautionary Tail: The Presence of an N-Terminal Tag
on Dynein Light-Chain Roadblock/LC7 Affects Its Interaction
with a Functional Partner Pp. 265-268
J. Song and J.L. Markley
[Abstract]
Biological Evaluation of Fluorinated p-Boronophenylalanine
Derivatives as a Boron Carrier Pp. 269-272
Y. Hattori, K. Kurihara, H. Kondoh, T. Asano, M. Kirihata,
Y. Yamaguchi and T. Wakamiya
[Abstract]
Radiolabeled Peptides and Proteins in Cancer Therapy
Pp. 273-279
C. Wängler, I. Buchmann, M. Eisenhut, U. Haberkorn
and W. Mier
[Abstract]
The Helical Structure Propensity in the First Helix
of the Histidine Phosphocarrier Protein of Streptomyces
coelicolor Pp. 281-290
E. Hurtado-Gómez, M. Caprini, A. Prieto and J.L.
Neira
[Abstract]
Genome-Wide Analysis of Enzyme Structure-Function
Combination Across Three Domains of Life Pp. 291-297
Z. Zhang and Y.-R. Tang
[Abstract]
T-Cell Antigen Receptor Assembly and Cell Surface
Expression Is Not Affected by Treatment with T-Cell Antigen
Receptor-Alpha Chain Transmembrane Peptide Pp. 299-302
N. Kurosaka, A. Bolte, M. Ali and N. Manolios
[Abstract]
Abstracts
[Back to top]
Influence of Conformationally Constrained Amino Acids
Replacing Positions 2 and 3 of Arginine Vasopressin (AVP)
and Its Analogues on Their Pharmacological Properties
D. Sobolewski, A. Prahl, I. Derdowska, A. Kwiatkowska,
J. Slaninová and B. Lammek
Synthesis of thirteen new analogues of arginine vasopressin
(AVP) has been described. Amino acid residues at positions
2 and 3 of AVP, [3-mercaptopropionic acid (Mpa)1]AVP
(dAVP), [Mpa1,D-Arg8]VP (dDAVP) and
[Mpa1,Val4,D-Arg8]VP (dVDAVP)
were replaced with one amino acid residue using sterically
constrained non-proteinogenic amino acids, 4-aminobenzoic
acid (Abz), cis-4-aminocyclohexanecarboxylic acid
(ach) or its trans-isomer (Ach). In the case of a
potent V1a
antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP,
only one similar analogue has been prepared by replacing positions
2 and 3 with Abz. Unfortunately, all new peptides were inactive
in bioassays for the pressor, antidiuretic and uterotonic
in vitro activities in the rat.
[Back to top]
Oligomerisation of Sarcoplasmic Reticulum Ca2+-ATPase
Monomers from Skeletal Muscle
D. Schreiber and K. Ohlendieck
The fast-twitch SERCA1 isoform of the sarcoplasmic reticulum
Ca2+-ATPase was purified to homogeneity and conjugated
to peroxidase. The SERCA1 probe showed high affinity binding
to the immobilized monomeric enzyme, but not crosslinker-stabilized
oligomers. This suggests a preferential complex formation
via homo-dimerization, rather than interactions with established
oligomeric structures.
[Back to top]
The Ways of Realization of High Specificity and Efficiency
of Enteropeptidase
A.G. Mikhailova, V.V. Likhareva, N. Teich and L.D. Rumsh
Comparative substrate analysis of full-length bovine enteropeptidase
and trypsin, bovine and human enteropeptidase light chains
was performed using model N-terminal dodecapeptides corresponding
to wild-type human trypsinogen and pancreatitis-associated
mutant trypsinogens K23R and D22G. The substitution of Lys
residue by Arg at P1 leads to 2-fold increase in the efficiency
of enteropeptidase hydrolysis; the absence of the negatively
charged residue at P2 reduces the efficiency of such hydrolysis
by two orders of magnitude. The difference in efficiency of
peptide chain hydrolysis after Lys/Arg residues by enteropeptidase
compared to trypsin is equal to the difference in hydrolysis
by serine proteases of different primary specificity of their
specific substrates.
[Back to top]
Stem Bromelain: An Enzyme That Naturally Facilitates
Oriented Immobilization
H. Khatoon, H. Younus and M. Saleemuddin
The lone oligosaccharide chain of stem bromelain was oxidized
with periodic acid to generate aldehyde groups and the resulting
oxidized enzyme coupled to amino-Sepharose in order to obtain
an immobilized preparation with uniformly oriented enzyme.
The immobilized bromelain exhibited high proteolytic activity
and remarkably enhanced thermal stability as compared to soluble
bromelain and that coupled to CNBr activated Sepharose.
[Back to top]
Critical Enzymes Involved in Endocannabinoid Metabolism
B.S. Basavarajappa
Investigations of the pathways involved in the metabolism
of endocannabinoids have grown exponentially in recent years
following the discovery of cannabinoid receptors (CB) and
their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol
(2-AG). The in vivo biosynthesis of AEA has been
shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase
D (NAPE-PLD), a secretory PLA2 and PLC. 2-AG, a
second endocannabinoid is generated through the action of
selective enzymes such as phosphatidic acid phsophohydrolase,
diacylglycerol lipase (DAGL), phosphoinositide-specific PLC
(PI-PLC) and lyso-PLC. A putative membrane transporter or
facilitated diffusion is involved in the cellular uptake or
release of endocannabinoids. AEA is metabolized by fatty acid
amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH
and monoacylglycerol lipase (MAGL). The author presents an
integrative overview of current research on the enzymes involved
in the metabolism of endocannabinoids and discusses possible
therapeutic interventions for various diseases, including
addiction.
[Back to top]
High-Level Expression and Purification of an Analgesic
Peptide from Buthus martensii Karch
Z. Cao, W. Wang, X. Xiao, K. Chen, X. Liang and D. Yu
BmK AngM1, a scorpion peptide isolated from Buthus martensii
Karch was reported to exhibit potential analgesic effect.
But the relative low content of this toxin in crude venom
limits its further characterization. In this study, we constructed
an expression vector and transformed into E.coli.
The BmK AngM1 was expressed as a fusion protein in the soluble
fraction and was purified by Nickel affinity chromatography.
Subsequently, the purified fusion protein was cleaved by enterokinase
and was further purified by reverse-phase HPLC. We purified
25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial
culture. The molecular weight of rBmK AngM1 determined by
ESI-MS was 7240.4 Da which was the expected size for correctly
processed. Analgesic bioassay studies of rBmK AngM1 exhibited
its potential analgesic effect comparable to that of the natural
BmK AngM1 peptide.
[Back to top]
Structural Insights into the Exchange Domain of Sec2p:
Expression, Purification, Crystallization, and Preliminary
X-Ray Diffraction Data Analysis
H.S. Gill
Sec2p is an essential yeast gene and is part of the cell polarization
process that leads to budding. The N-terminal domain of sec2p
(Sec2pN)?the guanine-nucleotide exchange factor for sec4p?has
been expressed in Escherichia coli, purified, and
crystallized. Crystals belong to the space group P21
with unit cell dimensions 178.1 x 98.4 x 180.0 Å, β
= 91.7o, and diffract synchrotron-generated X-rays to better
than 3.6 Å resolution. Pseudo-precession plots reveal
a Laue symmetry of 2/m, corresponding to the aforementioned
space group, and unusual weak diffraction in the ~5–7
Å resolution range. The Matthews number calculations
for a typical crystal density suggest a range of 28 to 64
molecules per asymmetric unit. Self-rotation and native Patterson
calculations demonstrate a pure helical array of protein subunits.
Based on the X-ray diffraction data analysis and amino-acid
sequence alignments, the paper presents a hypothetical model
of the exchange domain of sec2p as a pair of coiled-coil helices
that binds to sec4p and facilitates nucleotide disassociation.
[Back to top]
Design, Synthesis and Utilization of 1- Substituted
Sulphonyloxy- 2-Phenyl Benzimidazole as a Novel Peptide Coupling
Reagents
N.D. Kokare, R.R. Nagawade, V.P. Rane and D.B. Shinde
Highly efficient coupling reagents, N-methanesulphonyloxy-2-phenyl
benzimidazole and N - p-toluenesulphonyloxy-2-phenyl
benzimidazole were designed, synthesized and successfully
applied in peptide coupling reactions. Their efficiency was
evaluated by synthesizing a number of structurally different
amides and peptides as well. The distereomeric purity was
examined by HPLC. Also the optical rotations of all the synthesized
peptides were measured and found to be quite matching with
corresponding values in literature. After completion of reaction,
the N-hydroxy 2-phenyl benzimidazole which was the starting
material for the synthesis of reagents could be easily isolated
during the work up by acid base treatment and could be re-used
without significant loss in reactivity. Also the intermediate
in the reaction sequence was isolated and characterized by
mass and 1H NMR which could help to comment about
the probable mechanism.
[Back to top]
Cautionary Tail: The Presence of an N-Terminal Tag
on Dynein Light-Chain Roadblock/LC7 Affects Its Interaction
with a Functional Partner
J. Song and J.L. Markley
As part of structural investigations of components of the
molecular motor, dynein, we prepared the light chain, Robl1_mouse,
with and without an N-terminal His-tag. We found that the
His-tag introduced a spurious binding site for a second protein,
IC74. We propose a molecular mechanism for functional interference
by the His-tag.
[Back to top]
Biological Evaluation of Fluorinated p-Boronophenylalanine
Derivatives as a Boron Carrier
Y. Hattori, K. Kurihara, H. Kondoh, T. Asano, M. Kirihata,
Y. Yamaguchi and T. Wakamiya
Boron neutron capture therapy (BNCT) and magnetic resonance
imaging (MRI) are quite attractive techniques for treatment
and diagnosis of cancer, respectively. In order to develop
practical materials utilizing both for BNCT and MRI, fluorinated
p-boronophenylalanines and their alcohol derivatives
had already been designed and synthesized. In the present
paper the cytotoxicity, the incorporated amount into cancer
cells, and the tumor cell killing effects of these compounds
were elucidated to evaluate their usefulness as a boron carrier.
[Back to top]
Radiolabeled Peptides and Proteins in Cancer Therapy
C. Wängler, I. Buchmann, M. Eisenhut, U. Haberkorn
and W. Mier
With the advances in genomics, molecular biology including
gene vector technologies today’s molecular imaging modalities
have strongly been improved. The major progress is based on
peptide and antibody targeting vectors. When labeled with
β-emitting
radioisotopes these agents are applicable for endoradiotherapy
and exploit the targeting potential for highly specific therapeutic
applications. This novel class of pharmaceuticals offers the
potential to develop patient specific therapies and might
provide the means to go beyond the possibilities of current
chemotherapy and radiation therapy. In this review the basic
principles of endoradiotherapeutics based on peptides and
proteins are presented. Several of these drugs such as 90Y-rituximab
(Zevalin), 131I-tositumomab (Bexxar) and the somatostatin
receptor binding 90Y-DOTATOC that are nowadays
successfully applied in oncological therapy are discussed.
Future generations of endoradiopharmaceuticals will address
yet unknown targets which might be identified by screening
techniques such as ribosome and phage display peptide libraries.
[Back to top]
The Helical Structure Propensity in the First Helix
of the Histidine Phosphocarrier Protein of Streptomyces
coelicolor
E. Hurtado-Gómez, M. Caprini, A. Prieto and J.L.
Neira
The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase
system (PTS), formed by a cascade of several proteins, mediates
the uptake and phosphorylation of carbohydrates, and it is
also involved in signal transduction. Its uniqueness in bacteria
makes the PTS a target for new antibacterial drugs. These
drugs can be obtained from peptides or proteins fragments
able to interfere the first step of the protein cascade: the
phosphorylation of the HPr protein by the enzyme EI. We designed
a peptide comprising the active site and the first α-helix
of HPr of S. coelicolor; we also obtained a fragment
of HPr by protein engineering methods, comprising the first
forty-eight residues and thus, containing the amino acids
of the shorter peptide. Both fragments were disordered in
aqueous solution, with a similar percentage of helical structure
(~7 %), and an identical free energy of helix formation. In
40 % TFE, both fragments acquired native-like helical structure,
stabilized by non-native hydrophobic interactions, as shown
by the 2D-NMR assignments of the shorter peptide, and the
presence of similar NOE contacts in both fragments. These
findings, with the kinetic results in other members of the
HPr family, highlight the importance of short- and long-range
interactions during the folding reaction of HPr proteins.
Based on the residual helical population, hypothesis about
the inhibition capacity of the PTS by both fragments are discussed.
[Back to top]
Genome-Wide Analysis of Enzyme Structure-Function
Combination Across Three Domains of Life
Z. Zhang and Y.-R. Tang
To investigate diverse enzyme structure-function combination
(SFC) types in different species, 34 different genome sequences
were annotated using the protein catalytic domain database
SCOPEC (http://www.enzome.com/enzome/), in which both the
structure and function for each entry are known. Annotated
enzymes with catalytic domains from the same SCOP superfamily
are considered to have an identical structure. Annotated enzymes
sharing the identical three-digit EC number are considered
to have the same enzymatic function. Results reveal that the
different SFC types for enzymes identified in archaea, bacteria
and eukaryota are 137, 300 and 313, respectively. About 80%
of the SFCs identified in archaea can be consistently found
in bacteria and eukaryota species, whereas 28% and 35% combination
types in bacteria and eukaryota respectively are unique to
their corresponding groups. The number of functions per structure
and the number of structures per function for the annotated
sequences were measured in different species. Furthermore,
a new concept was proposed to represent enzymatic structures
as a functional similarity network. Thus, the current study
will be helpful to enhance the global view on the evolution
of enzymatic structure and function.
[Back to top]
T-Cell Antigen Receptor Assembly and Cell Surface
Expression Is Not Affected by Treatment with T-Cell Antigen
Receptor-Alpha Chain Transmembrane Peptide
N. Kurosaka, A. Bolte, M. Ali and N. Manolios
A synthetic peptide termed core peptide (CP), which corresponds
to a specific sequence of the TCR-α
chain transmembrane domain, is known to inhibit IL-2 production
in antigen stimulated T-cells. The molecular mechanism of
the TCR inhibition is not known. This study examined the effects
of CP on TCR subunit assembly and TCR cell surface expression
in vitro. Co-transfection experiments between TCR-a
and CD3-δ
using COS-7 cells, and the interaction between TCR-α
and the CD3 proteins in a T-cell line (2B4) were analysed
after incubation with CP or its conjugates. Results indicate
that CP co-precipitates with CD3-δ
and CD3-ε
in vitro, without any effect on TCR-α/CD3-δ
dimerisation or TCR multisubunit assembly and cell surface
expression.
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