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A ComputationalTool for Structural Biology: Crystallographic Refinement by Simulated Annealing
No full textConventional refinementof biological macromolecules involvesa series of steps, each of which
consists ofa few cyclesofrestrained least-squares refinement with stereochemical and internal
packingconstraints orrestraints that are followed by rebuilding the modelstructure with interactive
computer graphics. Duringthefinal stages of refinement solvent molecules are usually included
and alternative conformations for some atomsor residues in the protein may be introduced
Computer Simulations Applied to Site Specific Mutagenesis and Ligand Binding: The Use of Free Energy Perturbation Methods
No full textWegive a brief review ofthe theoretical methodology andfree energy perturbation
applications to proteins. We show that theoretical methods are capable of complementing
experimental studies in leading to a clearer understanding ofthe effect of site specific
mutations onligand binding, enzymecatalysis and protein stability
STRUCTURE-FUNCTION RELATIONSHIP IN PARATHYROID HORMONE
No full textParathyroid hormone (PTH) is the main regulator of calcium homeostasis and controls
resorption as well as formation of bone. We assayed overlapping PTH fragments for
their capability to stimulate either adenylate cyclase activity or DNA synthesis in
in appropriate renal or skeletal cell systems. From these studies we concluded that
PTH-induced enhancement of cellular cAMP levels and of cell proliferation are exerted
by different functional domains of the hormone molecule. Their conincidence with
structural domains will be discussed. After establishment of an efficient expression
system for human PTH in E. coli both functional and structural considerations lead to
a series of hormone variants. Their biological characterization showed that
individual PTH effects could selectively be affected by these mutations. Therefore,
site-directed mutagenesis conceivably opens up the possibility to select for either
anabolic or catabolic in vivo activities of PTH. Functional design of a potent
inducer-of bone growth might be of considerable clinical interest
DESIGN OF EFFICIENT HUMAN LEUKOCYTE ELASTASE INHIBITORS; VARIANTS OF HUMAN PANCREATIC SECRETORY TRYPSIN INHIBITOR (hPSTI)
No full textA set of hPSTI variants were constructed by total gene synthesis
or site-specific mutagenesis, with the aim of producing human
leukocyte elastase(HLE)-specific inhibitors and obtaining a better
insight into the parameters effecting inhibitor/protease interaction.
In the initial planning the structure of related Kazal-type
inhibitors were taken into account. For subsequent protein design,
models were made of HLE based on the structure of porcine pancreatic
elastase, and of hPSTI based on the stucture of porcine PSTI. Models
of the hPSTI/HLE and hPSTI/chymotrypsin complexes were generated by
CAPD "docking". The modelled complexes could be used to rationalise a
posteriori the inhibitory properties of the hPSTI variants, and to
postulate the structure of better elastase inhibitors which were duly
generated. Excellent specific inhibitors (Ky=1.5x107 4" M for SHEE)
were obtained and the contribution of individual amino acid residues
Or exchanges to the binding constant were estimated
IM RAHMEN DES BIOTECHNOLOGIE-PROGRAMMES VOM BUNDESMINISTERIUM FUR FORSCHUNG UND TECHNOLOGIE ZWISCHEN 1975 UND 1981 GEFÖRDERTE PROJEKTE ZUR PRODUKTION UND ZUR PRÜFUNG VON MIKROBIELLEM PROTEIN
No full textCOATS FROM MYXOCOCCUS XANTHUS: CHARACTERIZATION AND SYNTHESIS DURING MYXOSPORE DIFFERENTIATION
No full textAn extracellular coat from glycerol-induced myxospore of Myxococcus
xanthus has been isolated and characterized. Coats were examined chemically
and using both transmission and scanning electron microscopy. On a
dry weight basis, approximately 75 % of the coat is polysaccharide composed
entirely of galactosamine and glucose. The reminder of the coat
is protein (14 %), glycine (8 %) and organic phosphorus (less than 1 %).
Coats remained morphologically intact despite boiling in 10 M urea, sodium
lauryl sulfate plus B-mercaptoethanol, or extraction with warm
phenol. Coats also resisted digestion with a variety of proteolytic and
polysaccharide degrading enzymes. Synthesis of myxospore coat begins approximately
1 hour after the addition of glycerol to a culture. One
portion of the coat is complete by 5 - 6 hours but additional material
consisting primarily of glucose is added after 8 hours
BIOSYNTHESIS OF THE CAPSULAR MATERIAL OF MYXOCOCCUS XANTHUS
No full textPurified capsular material of Myxococcus xanthus has previously been
shown to be composed mainly of N-acetylgalactosamine, glucose and glycine.
Investigations carried out during the last two years have demonstrated
that UDP Gal NAc and UDP Glc are the major precursors. Biosynthesis of
capsule was studied by measuring the incorporation of 140 _upp Gal NAc,
344-UDP Glc and Su-glycine into TCA insoluable material. All three compounds
were incorporated more rapidly than into crude extracts prepared
from 2 hr glycerol induced myxospores that in similar extracts prepared
from vegetative cells. Preliminary studies indicated that incorporation
of UDP Gal NAc was stimulated by UDP Glc and to a lesser degree by glycine
and ATP. Similarly, incorporation of UDP Glc was stimulated by UDP
Gal NAc, glycine and ATP.
Both of the transferase activities were found exclusively in the 30,000
x g pellet, which were used for further studies. The glycine incorporating
activity remained in the supernatant. Both transferase activities were
linear with time for 1 hr and with protein concentration between
0.1 - 1.0 mg/ml. The incorporation of UDP Glc and UDP Gal NAc were absolutely
dependent upon divalent cations and were inhibited 40 % by 100 pg/ml
Bacitracin. In the absence of ATP, the incorporation of UDP Glc was stimulated
by UDP Gal NAc, but UDP Gal NAc incorporation was not stimulated
by various concentrations of UDP Gic. In both cases, the label was incorporated
into the 30,000 x g pellet, from which it could be recovered as
radioactive glucose and galactosamine following acid hydrolysis and paper
chromatography
MYXOSPORE INDUCTION IN STIGMATELLA AURANTIACA
No full textThe existence of different types of induction-resistant mutants allows
one to classify all known inducers into one of 3 groups; 1) the glycerol
group, 2) the phenethyl alcohol group, and 3) the t-butanol group. This
pattern, and the occurrence of specific antagonists which competitively
inhibit inducers of the glycerol group (e.g. oxindole, pyrrole), suqgest
that the inducers work by interaction with group-specific receptors on
the bacterial cell: receptor I for the glycerol aroup, receptor II for
the phenethyl alcohol group, receptor III for the t-butanol group of inducers.
There are only few inducers which may act on more than one receptor;
as isopropanol (receptors I+III), or indole (receptors I+II).
In order to become effective as an inducer, a compound needs an attachment
site which is responsible for its affinity to the receptor, and a
reaction site which determines the intrinsic activity of the inducer.
The reaction site of inducers of the phenethyl alcohol group may be the
aromatic ring, that of the glycerol group a positive charge or dipole
moment, for those are the only functional groups common to all members
of the respective groups. The conversion of t-butanol, which is a competitive
inhibitor of glycerol induction, into t-butylamine, which induces
at the glycerol receptor, seems to demonstrate that the amino
group is the reaction site of the molecule. Indeed, introduction of different
group-specific reaction sites into a certain molecule changes the
receptor specificity of the latter: t-butanol - receptors (I+) III;
phenylpropanol - receptor II;t-butylamine - receptors I.
The dose-response curves of myxospore induction are sigmoidal. This indicates
that induction is an all-or-nothing reaction. Inducers of different
groups if applied at concentrations which lie below the lowest
inducing concentration of each of them, add their individual effects.
This suggests that 1) the stimulus for myxospore induction is the same
with all types of inducers, and that 2) the stimulus has to reach a
threshold value before sporulation can occur.
It was possible to calculate the interaction of 2 different inducers,
or of an inducer and an inhibitor. The exact agreement between predicted
and experimentally determined effect indicates that our hypothesis and
of inducer-receptor-stimulus may come close to reality
MYXOBACTERIAL INTERACTION WITH PHIZOBIA
No full textThe predation of four soil myxobacteria - Myxococcus fulvus, “yxococcus
virescens, Myxococcus stipitatus and Polyangium fuscum on several susceptible
Rhizobium spp. was investiqated. No significant difference in
the course of attack on living and heat killed rhizobial cells was seen
in plate assays, but viable cells were resistant to the myxobacterial
culture filtrate in tube assays.
Some partially lysis-resistant rhizobia were rendered more susceptible
when separated from their polysaccharide. On hydrolysis, the purified
gum gave glucose, galactose, mannose and glucuronic acid; aminosugars
were absent. This qum had no effect on the synthesis and activity of lytive.
The partially purified lytic enzyme complex showed an N-acetyl
hexosaminidase activity on purified Rhizobium leguminosarum cell walls.
Viable cells, polysaccharide gum, purified cell walls and lipids ofR.
leguminosarum did not induce any chemotactic response in any of the
myxobacters tested, while autoclaved cells and cell proteins attracted
the migrating cells.
These myxobacters were also found in the rhizosphere of several local
legumes. When introduced in sterilized soil with added rhizobia,
M. fulvus and P. fuscum suppresses root nodulation
Marktdaten zur Biotechnologie : Produkte und Reaktoren
No full textAufgrund der hohen Wachstumsraten, die vielfach fiir den Biotechnologiemarkt
prognostiziert werden, und gefördert durch das Biotechnologie-Programm
der Bundesregierung, in dem die Entwicklung der Bioverfahrenstechnik
einen hohen Stellenwert einnimmt, kann man heute beobachten, daß zahlreiche
Anlagen- und Apparatebaufirmen erwägen, sich auf dem Gebiet der Biotechnologie
zu betätigen und zu investieren, um so langfristig an diesem Markt
teilzunehmen. Auch der Verband Deutscher Maschinen- und Anlagenbau eV
(VDMA) ist sich der wachsenden Bedeutung der Biotechnologie bewußt und
hat 1986 und 1987 Workshops zu diesem Problemkreis organisiert.
Allerdings differieren die Erwartungen und Prognosen über den zukünftigen
Biotechnologiemarkt außerordentlich stark. Selbst die Erfassung des derzeitigen
Marktvolumens ist schwierig. Die Diskrepanzen ergeben sich teilweise
durch unklare Definitionen darüber, welche Produkte der Biotechnologie
zugeordnet werden. Bei der Abschätzung des Potentials der neuen
Biotechnologieprodukte ist besonders deren Markteintritt unsicher. Viele
Prognosen sind spekulativ und nicht nachvollziehbar. "Bioprognostik"
stellt zur Zeit einen eigenen Markt mit nicht unbeträchtlichem Marktvolumen
dar. Nach einem Bericht von N. Rau in BTF-Biotech-Forum 3 (1986) 121 sind
über 300 Marktstudien, -analysen und -forschungsberichte über den Bereich
der Biotechnologie erhältlich, deren Preis sich aufsummiert auf etwa 2 Mio
DM beläuft. Für einzelne Studien sind bis zu 35.000 US $ zu zahlen, Seitenpreise
bis zu 100 DM sind keine Seltenheit. Die Vielzahl der Prognosen
allein schon verdeutlicht die Verwirrung.
Mit der Herausgabe dieses Hefts bemüht sich die GBF, die Diskussion zu
versachlichen, indem in Teil I - selbstverständlich ohne Anspruch auf
Vollständigkeit - Daten aus Marktstudien und Analysen zusammengestellt
wurden, auf deren Basis einige, wie wir meinen, konservative Schlußfolgerungen
über den erwarteten Bioboom gezogen werden können.
Im Teil II der vorliegenden Studie wird nur der Bioreaktormarkt betrachtet,
da viele Anlagen- und Apparatebauer, besonders die, die für die Pharma-,
Lebens- und Genußmittelindustrie tätig sind, hier einen günstigen Einstieg
in einen lukrativen Markt vermuten