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Papers by Dr. Susanne Billich

European Journal of Biochemistry, 1988

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a Igtl 1 ... more A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a Igtl 1 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by Bohmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)l. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities. Non-enzymatic 14-15-kDa proteins that bind fatty acids and fatty-acyl-CoA esters are abundant in the cytosol of cardiac tissues of mammals [2] including man 131. These 'cardiactype fatty-acid-binding proteins' (H-FABP), together with the fatty-acid-binding proteins of the hepatic (L-FABP) and intestinal (I-FABP) type, form, by the criteria of molecular mass and ligand binding, a group of related, yet structurally distinct proteins, whose functions are not well understood at present [2, 41. Presumed roles include (a) promoting the cellular uptake and intracellular utilization of fatty acids, (b) targetting fatty acids and acyl-CoAs to organelles, (c) modulating enzyme activities of lipid metabolism and (d) solubilizing fatty acids and acyl-CoAs in aqueous compartments. With oxidative fatty acid metabolism prevalent in heart cells, H-FABP has been inferred to take part in the delivery of fatty acids to mitochondria by a mechanism where a reversible self-aggregation of H-FABP in competition with its binding of fatty acids regulates the availability of fatty acids for /?-oxidation [5]. Glatz et al. observed a diurnal variation of H-FABP contents in rat heart cells, from 4% of cytosolic proteins at noon to 8% at midnight, that correlated with palmitate oxidation [6]. After immunoassays and cDNA probes for ratfatty-acid-binding proteins became available, it was shown

Antiviral Chemistry and Chemotherapy, 1992

The integration protein of the human immunodeficiency virus type 1 was purified from recombinant ... more The integration protein of the human immunodeficiency virus type 1 was purified from recombinant bacteria overproducing this enzyme. The final step of purification, namely chromatography on polyUsepharose, yielded a homogeneous protein preparation showing specific DNA cutting and joining activities. For a convenient assay of the endonuclease reaction, a 21-mer duplex oligonucleotide corresponding to the U5-LTR end of the viral DNA was radiolabeled at the dinucleotide that is removed by the enzyme. After the reaction, assay mixtures were passed through DEAE-cellulose filters which bind the substrate, but not the short radiolabeled product. Thus, an enzyme-dependent decrease of bound radioactivity was observed with time. Reaction rate was linearly dependent on enzyme concentration and the amount of substrate used was far below saturating concentrations. The reaction showed a pH-optimum at 7.5 and was strictly dependent on the presence of Mn2+. The presence of reducing agents like 2-me...

Antiviral Chemistry and Chemotherapy, 1991

Antiviral chemistry & chemotherapy

Biological chemistry Hoppe-Seyler

The EMBO Journal

Communicated by K.Moelling Retroviruses code for a specific protease which is essential for polyp... more Communicated by K.Moelling Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pot gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence-but also conformationdependent. This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lamda g... more A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lamda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted.
H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous
to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by Boehmer et al. [J. Biol. Chem. 262, 15137- 15143 (1987)l. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.

The integration protein of the human immunodeficiency virus type 1 was purified from recombinant ... more The integration protein of the human immunodeficiency
virus type 1 was purified from recombinant
bacteria overproducing this enzyme. The final step of
purification, namely chromatography on polyUsepharose,
yielded a homogeneous protein preparation
showing specific DNA cutting and joining activities.
For a convenient assay of the endonuclease reaction,
a 21-mer duplex oligonucleotide corresponding to the
U5-LTR end of the viral DNA was radiolabelled at the
dinucleotide that is removed by the enzyme. After the
reaction, assay mixtures were passed through DEAEcellulose
filters which bind the substrate, but not the
short radiolabelled product. Thus, an enzyme-dependent
decrease of bound radioactivity was observed
with time. Reaction rate was linearly dependent on
enzyme concentration and the amount of substrate
used was far below saturating concentrations. The
reaction showed a pH-optimum at 7.5 and was strictly
dependent on the presence of Mn2+. The presence of
reducing agents like 2-mercaptoethanol was not
essential for enzymatic activity. The assay was used to
test selected compounds for their inhibitory potential
against integrase. Typical inhibitors of DNA-topoisomerases
did not inhibit the endonuclease reaction,
with the exception of the intercalative agent actinomycin
D which blocked the reaction with an IC50-value
of 3µM. Dextran sulphate inhibited the enzyme with an
IC50 = 1.6µg/ml.

Retroviruses code for a virus-specific protease which is essential for polyprotein processing and... more Retroviruses code for a virus-specific protease which
is essential for polyprotein processing and antiviral infectivity.
The human immuned eficiency virus- 1 protease
is an aspartic protease 9o fk Da whichw as synthesized
by recombinant DNA technology and arises by autocatalytic
processing from a polyprotein precursor
which has recently been demonstrated by use of a
protease-specific monoclonal antibody. The protease
was shown to form dimers. Here we demonstrate that
synthetic peptides can be used as both models ubstrates
as well as inhibitors for investigation of the protease.
14 synthetic peptides, 7-18 amino acids in length,
containing putative protease cleavage sites of the viral
polyprotein gag and pol precursors, have been analyzed
with the partially purified protease by the use of
high performance liquid chromatography. In seven
cases, where cleavage was observed, the length of the
peptides did not significantly influence the cleavage
efficiencies, heptapeptides being large enough as model
substrates. No cleavage was observed with a protein
preparation purified in parallel from control bacteria
not expressing the human immune deficiency virus-1
protease. The protease was not only able to cut next to
a proline but also between other peptides indicating
that the proline is not a prerequisite. Three peptides
with either reduced bonds at the cleavage site or a
substitution by statin were inhibitory while another
uncleaved substrate was not. The usefulness of small
model substrates for characterization of the protease
is further demonstrated by determination of kinetic
optimum pH (3.6-6.6) and incubation temperature
(37 “C).

J. Biol. Chem. , 1988

virus-specific protease which is essential for polyprotein processing and viral infectivity. The ... more virus-specific protease which is essential for polyprotein processing and viral infectivity. The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody. The protease was shown to form dimers. Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease. 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography.

Retroviruses code for a specific protease which is essential for polyprotein precursor processing... more Retroviruses code for a specific protease which is essential
for polyprotein precursor processing and viral infectivity.
The HIV-specific protease has been predicted to
be an aspartic protease which is located at the amino terminus
of the pot gene. We have prepared several constructs
for bacterial expression of the protease. Two of
them span the whole protease region and result in its
autocatalytic activation. Analysis of the dynamics of this
activation indicates a two-step process which starts at the
carboxy terminus and ends at the amino terminus of the
protease. The activated protease is a molecule of 9 kd
as evidenced by monoclonal antibody in immunoblot
analysis. A construct in which the carboxy terminus of
the protease is deleted results in a stable, enzymatically
inactive 27-kd protein which proved useful as substrate
since it contains one of the predicted cleavage sites. The
stability of this protein indicates that the carboxy-terminal
sequences of the protease are essential for its activity and
its autocatalytic activation. The protease which is very
hydrophobic was solubilized by acetone treatment and
passaged over ultrogel and propylagarose columns for
partial purification. It elutes as a dimer and tends to
aggregate. It is inhibited by pepstatin A in agreement with
its expected active site and its theoretical classification
as aspartic protease. Cleavage of the gag precursor results
in the mature capsid protein, p17. The protease does not,
however, cleave the denatured 27-kd substrate or the
denatured gag precursor. Therefore its specificity appears
to be not solely sequence- but also conformationdependent
. This property needs to be taken into account
for the development of protease inhibitors for therapy
of AIDS.

European Journal of Biochemistry, 1988

Antiviral Chemistry and Chemotherapy, 1992

Antiviral Chemistry and Chemotherapy, 1991

Antiviral chemistry & chemotherapy

Biological chemistry Hoppe-Seyler

The EMBO Journal

The integration protein of the human immunodeficiency virus type 1 was purified from recombinant ... more The integration protein of the human immunodeficiency
virus type 1 was purified from recombinant
bacteria overproducing this enzyme. The final step of
purification, namely chromatography on polyUsepharose,
yielded a homogeneous protein preparation
showing specific DNA cutting and joining activities.
For a convenient assay of the endonuclease reaction,
a 21-mer duplex oligonucleotide corresponding to the
U5-LTR end of the viral DNA was radiolabelled at the
dinucleotide that is removed by the enzyme. After the
reaction, assay mixtures were passed through DEAEcellulose
filters which bind the substrate, but not the
short radiolabelled product. Thus, an enzyme-dependent
decrease of bound radioactivity was observed
with time. Reaction rate was linearly dependent on
enzyme concentration and the amount of substrate
used was far below saturating concentrations. The
reaction showed a pH-optimum at 7.5 and was strictly
dependent on the presence of Mn2+. The presence of
reducing agents like 2-mercaptoethanol was not
essential for enzymatic activity. The assay was used to
test selected compounds for their inhibitory potential
against integrase. Typical inhibitors of DNA-topoisomerases
did not inhibit the endonuclease reaction,
with the exception of the intercalative agent actinomycin
D which blocked the reaction with an IC50-value
of 3µM. Dextran sulphate inhibited the enzyme with an
IC50 = 1.6µg/ml.

J. Biol. Chem. , 1988