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A Consensus Match Scoring System thatis Correlated with Biological Functionality
No full textThe C;-scoring system is suitable for the identification of putative functional transcription factor
binding sites solely by sequence analysis. This is a very important feature since it allows
preselection of candidate bindingsites for experimental analysis from uncharacterized genomic
sequences. Data derived from the analysis of almost 2.4 million nucleotides of genomic sequences
show a good correlation of C;-scoring with known biological function. High-scoring bindingsites
are clearly over-represented in putative control regions of the genomic sequences. Known
functional bindingsites cluster in the same regions. Furthermore, we demonstrate high C;-scores
to correlate with biological functionality of 26 individual binding sites for three completely
unrelated transcription factors. Consensus matches knownto be either nonfunctional or to bind
their correspondingprotein factors with highly reduced affinity are low-scorers in ourrating
Classification of Local Protein Structural Motifs by Kohonen Networks
No full textKohonen networks were used for automatic classification of local structural elements derived
from a set of 136 non-homologousproteins. A reduced representation of protein backbones based
on dihedral phi and psi angles was employedfor construction of training patterns. Segments of
nine residues were transformed into a 16-dimensional description by their angular values.
Kohonen-mapping yielded a two-dimensional topological map of representative structural motifs.
Helical structures and sheets are located on opposite sides of the feature map, and several
intermediate forms are found. The map wasusedto trace protein backbones of two proteins,
cytochrome bs and y-IV crystallin, leading to characteristic trajectories in the feature map
THE USE OF FLUORESCENCE SENSORSAS OPTICAL BIOSENSORS
No full textThe use of miniaturized fluorescence probes for on-line monitoring analytes in continuous-
flow systems (automated clinical analysis, process control, e.g. in biotechnological
processes, environmental control, etc.) has becomeincreasingly important. Wereport
the application of a fiber-optic based fluorescence probe used as optrode for a novel
optical biosensor system to monitor NAD(H) dependent enzymatic reactions based on the
flow-injection analysis (FIA) principle. A miniaturized fiber-optic fluorimeter is combined
with a small size membrane reactor (1 ml) (Fig. 1). In here, enzymes (dehydrogenases)
for analyte conversion and coenzymeregeneration as well as the macromolecular
coenzyme derivative polyethyleneglycol (PEG, M, = 20,000)-N°-(2-aminoethyl)-NAD(H)
[1] are retained in the direct vicinity of the sensor tip of a concentric fiber-optic probe
(fluorescence excitation 360 nm / emission 460 nm) by an ultrafiltration membrane (cutoff:
5000 Dalton) (Fig. 2). Only low molecular-weight compounds (analytes, products,
etc.) can freely pass through thisultrafiltration membrane
ONLINE MONITORING OF L-LYSINE IN TECHNICAL CHROMATOGRAPHY BY FLOW INJECTION ANALYSIS(FIA)
No full textAn enzymatic assay is presented which is highly selective for L-lysine and‘is based on FIA
techniques combined with photometric detection. L-Iysine-a-oxidase (EC 1.4.3.14) from
Trichodermaviride and horseradish peroxidase were coimmobilized on VA Epoxy resin in
an analytical enzyme reactor, which wasincorporated in the FIA system. 30 samples hot,
sample volume2 ul, linearity range 1-16 mM L-lysine, correlation coeficient 0.998, limit of
detection 1 mM and 0.5 % RSD. The enzyme Cartridge can be used for several thousand
measurements over a period some of several months. The methodis applicable for monitoring
in technical ion exchange chromatography. The values correlate well with the ones
determined with an amino acid analyser. Breakthrough point detection is possible and productspecific
control may now be achieved
DEVELOPMENT OF ONE-SHOT FLOW INJECTION ANALYSIS WITH IMMOBILIZED ENZYME COLUMN AND ITS APPLICATION TO CLINICAL ANALYSIS
No full textThe availavility of enzymes as the reagents for clinical analysis has
been wellknown. The enzymes can be used not only in the form of solution
but also in the form of immobilized enzyme reactor. The determinations
of glucose and uric acid in serum by chemiluminometry after
the production of hydrogen peroxide by the respective oxidase are presented.
Newly chemiluminometric methods for ammonia and NAD(P)H using
an enzyme column reactor consisting of both immobilized L-glutamate
dehydrogenase and L-glutamate oxidase, and its application to other
enzymatic analyses that give ammonia and NAD(P)H as a final signal, e.g.
urea and magnesium, are described. A compact automated analyzer which
can analyse constituents in biological fluids using these enzymatic
methods with a small sample volume and in a short time has been developed.
The instrument is composed of an one-shot FIA system, a FIA
system of new type, equipped with chemiluminometric detection and a
immobilized enzyme column reactor used in combination. The one-shot
FIA system is also described
STEREOSELECTIVE DETERMINATIONOF L- AND D-AMINO ACIDS IN SERUM BY THEUSE OF IMMOBILIZED ENZYME REACTORSIN FIA
No full textA flow injection method containing immobilized enzyme reactors (IMERs) ; L-amino acid oxidase
(L-AAO), D-aminoacid oxidase (D-AAO)and Horse radish peroxidase (HRP) for the determination of
L-and D-amino acids has be en developed. Enzymatic conversion efficiencies for twenty L-and D-amino
acids are given. The linear range of the detecteable amino acids was around two orders of magnitude. The
sample frequency was 19 h” with a sample volume of 50 yl. The methodis used for the stereoselective
determination of L-and D-aminoacids in the presence of L-aminoacids in serum
APPLICATIONS AND KINETICS OF IMMOBILIZED ENZYMES AND COUPLED ENZYME REACTIONS
No full textImmobilization of enzymesresults in more adequate reagentsto be used analytically. After immobilization,
the kinetic parameters of the enzymes are modified and nothing can be predicted abouttheactivity of
the heterogeneous system. In coupled enzymereactions with co-immobilized enzymes, this is even more
important owingto the kinetic dependence on each consecutive reaction. The determination of ethanol and
acetaldehyde is considered in this paper, using two different coupled enzyme systems. Some important
parameters, as the enzyme charged,the ratio of each enzymein the sequence and the immobilization yield,
are considered in terms of conversion efficiency finally determiningthesensitivity of the analysis
Exchangeable Immobilized Enzyme Reactor (EIMER)
No full textSince Flow-Injection Analysis (FIA) was introduced by Ruzicka and Hansenin 1975, a
large numberofapplications using biological active reactors have been discribed. These
reactors are often designedas fixed bed reactors with bio-material held between two nets
or immobilized in tubes or on membrane surfaces. These designs allow the use of the
enzyme’s naturalcatalytic activities. When denaturation, fowling or inhibition occure,
however, thereislittle chance of reactivating the enzyme and the entire reactor must be
discarded.
Ourintent to design a fully automated biosensor for pesticide detection using enzyme
inhibition made it necessary to develop methods for exchanging immobilized enzyme
between two runs of analysis
Processing and Re-Processing of Asparagine-linked Oligosaccharides
No full textThe assembly of asparagine-linked oligosaccharides in glycoprotein biosynthesis is cell specific,
polypeptide specific and glycosylation site specific. Recombinant glycoproteins produced in nonhomologous
cells are likely to be glycosylated abnormally and the consequences on proteinstability,
conformation and biological activity need to be considered. Although the major pathways of assembly
of asparagine-linked oligosaccharides are identified, their regulation during biosynthesis is not
understood. The early events in oligosaccharide processing catalyzed by glucosidases I and II and
specific mannosidases are particularly complex. Experiments using various inhibitors of processing
glucosidases and mannosidases as well as structural analysis of processing intermediates, show that
different processing pathways. are selected for assembly of glycans substituted at specific sites in
glycoproteins. New mannosidases are being described that participate in these diverse pathways. A
novel mannosidase of rat liver is concentrated in endosomes as well as the cis Golgi compartment and
may play an additional role in remodelling of glycoproteins that occurs during internalisation and
recycling of cell surface glycoproteins
PANCREATIC LIPASE : CRYSTALLIZATION, DOMAIN INVESTIGATION AND CLONING
No full textPancreatic lipase is involved in dietary fat digestion. The
enzyme, which belongs to a special class of esterases, realizes
an heterogeneous catalysis. Although extensively studied for
many years, the mechanism of action of lipase on emulsified
substrates is still not elucidated.
Crystals of man and horse pancreatic lipases suitable for an
X-ray investigation have been obtained. The cell parameters of
the various crystalline forms have been determined. Heavy atom
derivatives are under investigation.
The spatial organization of pancreatic lipase in two well
defined domains has been studied through limited proteolysis of
the enzyme from various species.
A horse pancreatic cDNA library has been built and the cDNAs
encoding for lipase and colipases isolated