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Biomedical subjects

R Frank

Publications and source records attributed to R Frank.

At least 289 records · Page 16Linked to original sources
PubMed1990

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method.

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.

Animals↗
PubMed1990

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung. Homology between the two subunits of the enzyme.

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77,500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Amino Acid Sequence↗
PubMed1990

Human cytosolic adenylate kinase allelozymes; purification and characterization.

Cytosolic adenylate kinase (ATP + AMP in equilibrium 2 ADP:AMP phosphotransferase, EC 2.7.4.3) shows a balanced polymorphism of AK1*1 (95%) and AK1*2 (5%). AK1*2 occurs only in Caucasian populations and is common among hemophilia-A patients (László, A., Kósa, F., Zimányi, I. and Egyed, A. (1985) Acta Paediatr. Hung. 26, 87-96). 1. From 350 g heterozygous human muscle we purified 7.5 mg AK1*2 and 8.5 mg AK1*1. The final step was the separation of the two allelozymes by preparative cellulose acetate gel electrophoresis. Throughout the preparation, the AK1 allelozymes were stabilized by adding diadenosine pentaphosphate. When necessary the proteins were freed from this ligand using phosphocellulose chromatography at pH 5.2. The specific activity of AK1*2 (420 U/mg protein at 25 degrees C) is 3.5-times lower than that of AK1*1, whereas the Michaelis constants do not differ for the allelozymes. The molecular mass of both proteins was found to be 21.7 kDa. 3. High-performance liquid chromatography of tryptic peptides revealed no differences between AK1*1 and AK1*2 except for the peptide t(109-127). Sequence analysis showed that Glu-123 in AK1*1 is exchanged for Gln-123 in AK1*2. In light of the known three-dimensional structure of AK1*1, this substitution alone might account for the different properties of the allelozymes.

Adenylate Kinase↗
PubMed1990

Structure and biosynthesis of the signal-sequence receptor.

The signal-sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino-terminal signal sequence and contains only one classical membrane-spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal-recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell-free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy.

Amino Acid Sequence↗
PubMed1990

NMR and molecular dynamics studies of the mKr2 'zinc finger'.

We determined the structure of a 30-amino-acid 'zinc finger' motif from the mouse Kruppel-like gene mKr2 in solution in the absence of metal ions by two-dimensional NMR, distance geometry and restrained molecular dynamics methods. The most prominent secondary structural feature of the peptide is a helix extending from Ser14 to Ile20. The zinc-containing structure of the peptide was simulated by molecular dynamics calculations with restraints derived from the known geometry of the zinc ion and the ligating amino acid residues Cys6, Cys9, His22, His26. The latter structure does not deviate markedly from the set of structures determined for the zinc-free peptide, i.e. this peptide can accommodate a Zn2+ ion without major structural rearrangements. Thus, we propose that the metal ion stabilizes the existing three-dimensional peptide structural motif rather than introducing novel structural features.

Amino Acids↗
PubMed1990

The head dome: a simplified method for human exposures to inhaled air pollutants.

Acute controlled exposures of human subjects to air pollutants are customarily carried out with whole-body chambers, masks, or mouthpieces. The use of these methods may be limited by cost or technical considerations. To permit a study involving a highly unstable pollutant, artificial acid fog, administered to subjects during natural breathing, a head-only exposure chamber, called a head dome, was developed. It consists of a transparent cylinder with a neck seal which fits over the subject's head and rests lightly on his shoulders. The head dome does not constrain the upper airways or impede exercise on a bicycle ergometer. Ventilation can be monitored accurately and unobtrusively with a pneumotachograph at the exhaust port of the dome. A thermocouple may be used to monitor the onset and persistence of oronasal breathing. For short-term exposures to unstable or reactive pollutants lasting up to several hours, the head dome is an effective alternative to a whole-body chamber and probably superior to a face mask or mouthpiece.

Air Pollutants↗
PubMed1990

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.

Adenosine Triphosphatases↗
PubMed1990

Solution structure of the DNA-binding domain of the yeast transcriptional activator protein GCN4.

The solution structure of an active synthetic peptide containing both the leucine zipper and the adjacent basic domain of the yeast transcription factor GCN4 (residues 220-280) was determined by NMR. The two domains show structurally distinct behaviours. In the absence of DNA, the basic domain is, although very flexible, structured and fluctuating around a helical conformation. The leucine zipper region forms a long, uninterrupted helix. From a suitable set of NMR distances the three-dimensional structure of the leucine zipper monomeric sub-domain was calculated by distance geometry algorithms. The structure of the symmetrical parallel dimer was obtained by model building using the NMR information. A smaller peptide with the sequence of the isolated basic region (residues 1-35 of the 61 residue peptide) was also synthesized. Circular dichroism studies showed 30-40% helicity. A flexible helix spans the region between residues 8 and 21. The comparison of our results with suggested models is discussed in detail.

Algorithms↗
PubMed1990

Identification of seroreactive regions of the human papillomavirus type 16 protein E4, E6, E7 and L1.

Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid. Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants. Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and L1 (three regions) open reading frames could be found by this approach. Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids. In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum. Using an approach to predict 'receptor-like' regions within the respective proteins, five of the seven regions were also identified. From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA.

Amino Acid Sequence↗
PubMed1990

Better predictors of successful His-bundle ablation analysis of first shocks.

We have reported here that a longer HV interval in association with a larger His amplitude yields a high rate of success when used to position the ablating catheter for His-bundle ablation. Additionally, we have shown that double discharge shocks are more effective than single discharge shocks, and that negative polarity is more effective than positive polarity. The use of bipolar or tripolar, and not quadripolar catheters, was also associated with a higher success rate. In our institution, using a bipolar catheter, we attempt to record an HV interval greater than 55 msec and a His amplitude greater than 0.35 mV. When both of these criteria are fulfilled, we use 3 to 4 joules per kg, and a single discharge shock. When one or the other of these criteria are not fulfilled, we use the double discharge shock method. Using these techniques, we have achieved successful His-bundle ablation with only one shock in all but one of the most recent 21 consecutive patients.

Bundle of His↗
PubMed1990

The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

Amino Acid Sequence↗