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THE USE OF BIOSENSORS IN DETERMINATION OF DIFFERENT SUBSTANCES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
No full textA rapid, simple and economic method was developed, which combines the specificity
of enzymes with the high sensitivity of HPLC. Therefore sample pretreatment is reduced
to simple dilution or extraction steps.
In this work oxidases for alcohol, glucose, oxalic acid, ascorbic acid and galactose
were immobilized and used as biosensors. Food samples were fruit juices and vegetable
products, soft drinks and wine. The detection limit e.g. for oxalic acid was in the
picomol range
Ethanol fermentation monitoring by enzymatic Multichannel-FIA
No full textA four channel FIA-System was developed for the online process monitoring of an
anaerobic bacterial ethanol fermentation with Zymomonas mobilis in a fluidized bed
reactor. Glucose, ethanol, acetaldehyde and L-lactate are determined by fluorimetric
detection of enzymatically formed NADH. The principle of synchronized merging
zone was combined with paralel configurated gas diffusion and dialysis. Membran
separation serves for the elimination of proteins and proteases and for the dilution
of the analytes. The FIA equipment is connected to the fluidized bed reactor with a
crossflow microfiltration module. By means of an intelligent valve configuration the
measuring device is controlled by an user-friendly software running on a personal
computer. All steps of the process analytical regime i.e. detection, calibration,
recalibration and calculation of the analytical results are computer controlled
Design and Application of a new Two-Channel FIA
No full textA new two-channel pneumatic slider valve was constructed by modification of a commercial
valve. This valve simplifies the flow injection analysis of two components in a sample stream
utilizing general advantages of pneumatically driven slider valves as there are small sampling
volume(22 ul), high frequencies of analyses (25 sec), simple construction and maintenance, high
reliability, low costs and,finally, ease of multiplication, as many slider valves can be operated by
one pneumatic actuator, if installed in an appropriate housing.
The performanceofthe valve is exemplified by analysis of a gelfiltration with penicillin
acylase, alkaline phosphatase and molecular weight markers. The new designed valve, integrated
in FIA systems, enables the independentanalysis of two different components or, alternatively,
the analysis of one componentby different assays
SMALL MOLECULES AS PROBES FOR THE SECRETORY PATHWAY
No full textMarkers for the unsignalled vesicular flow, the "bulk flow",
from the Endoplasmic Reticulum (ER) and the Golgi apparatus were
generated according to the following concept: A substrate analog
that is able to diffuse through biological membranes is added to
living cells. This substrate can be converted into a derivative
that is unable to permeate membranes by a corresponding enzyme
that is uniquely located to the organelle from which vesicular
bulk flow is to be measured. If the non diffusable derivative
reaches the medium of the cell culture, it must have been
transported in the luminal side of vesicles, thus serving as a
bulk phase marker. By use of diffusable substrate analogues that
are radioactively labeled the kinetics of secretion of such bulk
phase markers can easily be quantitated. Two examples for this
approach are described
ISOLATION OF GLYCOPEPTIDES OF THE ENVELOPE GLYCOPROTEIN FROM A POLYTROPIC MURINE RETROVIRUS
No full textA polytropic recombinant retrovirus containing the envelope gene of
Friend mink cell Focus-inducing virus plus the remainder of the genome
of an amphotropic murine leukemia virus was propagated on mink lung
cells in the presence of F222 Hterenninaes Radiolabeled viral glycoprotein
was isolated hy immunoaffinity chromatography and purified by
preparative SDS polyacrylamide gel electrophoresis. Glycopeptides obtained
after digestion with trypsin were fractionated by gel filtration
and reversed-phase HPLC, and Oligosaccharides attached were characterized
by their sensitivity towards endo-ß-N-acetylglucosaminidase H
and glycopeptidase F and, in part, by chromatographic Procedures and
methylation analysis.
The results obtained Provide evidence for a specific glycosylation
of the viral glycoprotein in mink lung cells with Tegard to the distribution
of high-mannose and Complex type N-glycans
INCORPORATION OF FUCOSE INTO CHICK BRAIN GLYCOPROTEINS: EFFECTS OF THE AMNESIC AGENT 2-DEOXYGALACTOSE
No full textThe amnesic agent 2-deoxygalactose inhibited total exogenous [}4c}fucose incorporation into chick
forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic analysis
showedthat intracerebrally injected [3H]2-deoxygalactose labelled the same eight major glycoprotein
components as were identified by [4Cjfucose labelling. Between 4 and 24 hours after administration
of [3H]2-deoxygalactose, the incorporated radioactivity was found predominantly at the synapticsites,
some glycoproteins being more abundant in synaptic plasma membranesandothers in postsynaptic
densities. The pattern of distribution varied according to the timeafter injection. One-trial passive
avoidance training of chicks decreased fucose uptake into components of molecular mass 150-180
kilodaltons butsignificantly increased its uptake into glycoproteins of molecular mass 33 kilodaltons
and 28 kilodaltons
GLYCOSYL PHOSPHATIDYLINOSITOL ANCHORS
No full textThe earliest studies suggesting proteins were associated with membranes solely through a
covalently-linked glycosyl phosphatidylinositol (GPI) took advantage of Pl-specific phospholipases
(PIPLCs) [reviewed by Low (1)]. The demonstration in the mid-1970s that alkaline phosphatase
could be released from tissues by PIPLCs purified from B. cereus (2) and S. aureus (3) led
eventually to the suggestion (4) of a direct protein-PI linkage. Another line of evidence, which
suggested that the hydrophobic properties of a protein could be due solely to the covalent,
C-terminal attachment of a lipid moiety containing ethanolamine and glucosamine, came from the
protein sequence and compositional analyses of Thy-1 published in 1981 (5). It was not until
1988, however, that the complete primary structure of the GPI anchor of Thy-1 was published (6).
Publication in the same year (7) of the complete structure of the Trypanosoma brucei variant
surface glycoprotein (VSG) anchor culminated a long series of investigations that also began in the
early 1980s (see ref. 8) Another long-standing investigation, that of human acetylcholinesterase
(AChE), has led to the recent publication of a third protein-linked GPI structure (9,10). In fact,
the last five years have witnessed an explosion in our knowledge of GPls, including not only their
Structural features, but also a proposed biosynthetic pathway and information from gene fusion
experiments about the signals directing their addition to proteins and their functional importance
Methods Suitable for Large Scale Quantitation of Sugar Residues in Microheterogenic Glycoproteins
No full textThree methods suitable for the purpose of large scale quantitation of sugar residues in samples of
microheterogenic glycoproteins are presented, named by the generic names 1) Marked-Lectin
Immunoassay (MALIA), 2) Oxidative-Reductive Immunoassay (Redox-IA), or 3) relying on
incorporation of labeled sugars from precursors using serum glycosyltransferases. All three methods
can be used in the presence of serum and comprise only simple incubations and washings that would be
possible to perform with standard or slightly modified RIA or ELISA equipment.
1) In MALIA, the glycoproteins are bound to an immunoadsorbentandthis is used in combination
with an incubation with marked lectin. The utility of the method is restricted only by the specificity of
the lectin and its affinity for the solid phase antigen during washing. When thelectin has low affinity
for the sugar residues, they can be measured in the washing supernatants with a similar sensitivity of
method.
2) In Redox-IA, the contentofsialic acid in a glycoprotein bound from a body fluid onto an
immunoadsorbent is measured using the increased Susceptibility of sialic acid residues to oxidation
which are a) oxidized with prefered specificity and b) reduced using a labeled reductant followed by c)
isolation of the labeled species. The utility of the method is restricted to measurementsof averagesialic
acid content in glycoproteins whose identity is specified by their affinity to the immunoadsorbent.
3) Using the endogenous serum glycosyltransferases and labeled nucleotide-linked sugars, crude
information can be obtained about the stage of glycosylation of the serum components. The previously
predominant method to precipitate the labeled species with acid has been replaced by a method using
solid phase lectins onto which the labeled product is collected with preserved immunospecificity
STEREOSELECTIVITY OF LIPASES: Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique
No full textIn the present study, porcine pancreatic lipase (PPL), rabbit gastric lipase (RGL) and humangastric lipase (HGL)
stereospecificity towards enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film
technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1 ,2)-diacyl-sn-glycerol or enantiomeric 1(3)-alkyl-2-acyl-snglycerol
or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase
stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The
cleaved acyl moiety was the samein both enantiomers, thereby excluding the possibility of effects occuring dueto fatty acid
specificity. We observed a PPL sn-3 stereoselectivity when using the enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol
(diglyceride analogue) which contrasted with the lack ofstereoselectivity observed when using the enantiomeric 1(3)-alkyl-
2,3(1,2)-diacyl-sn-glycerol (triglyceride analogues). The gastric lipases, in contrast to the pancreatic lipase, preferentially
catalyse the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoalky] diacylpairtested.
From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of RGL, whereas low
rates and a clear chiral discrimination was noticed in the case of HGL duringcatalysis of the acyl chain from the secondary ester
bond of 1(3)-alkyl-2-acyl enantiomers.It is particulary obviousthat in the case of HGL decreasing the lipid packing increases
the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylaminoderivatives
(diglyceride analogues)
STEREOSELECTIVITY OF LIPASES : Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases
No full textIn the present study, porcine pancreatic lipase (PPL), rabbit gastric lipase (RGL) and human gastric lipase (HGL)
stereospecificity towards chemically like, but sterically non equivalent ester groups within onesingle triglyceride molecule was
investigated. Lipolysis reactions were carried out on synthetic trioctanoin ortriolein, which are homogenous, prochiral
triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were
derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further
separated by HPLC. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases
stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The
stereoselectivity of HGL and RGL,expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74%
and 47% fortriolein, respectively. The corresponding values with PPL were 3% in the caseof trioctanoin and 8% in that of
triolein. It is worth noting that RGL, unlike HGL, became morestereoselective for the triglyceride with shorter acyl chains
(trioctanoin). This is one of the moststriking catalytic differences observed between these twogastric lipases