Biomedical subjects

D Kaloyanova

Publications and source records attributed to D Kaloyanova.

4 recordsLinked to original sources

PubMed2001

Sphingomyelin-enriched microdomains at the Golgi complex.

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Adenosine Triphosphatases↗

PubMed1999

Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

Animals↗

PubMed1998

The low expression level of pokeweed antiviral protein (PAP) gene in Escherichia coli by the inducible lac promoter is due to inefficient transcription and translation and not to the toxicity of the PAP.

Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase which inactivates both eukaryotic and prokaryotic ribosomes. Due to this activity the wild-type PAP gene encoding mature protein has not so far been expressed in Escherichia coli. In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E. coli under an inducible lac promoter. In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E. coli cells to produce a much higher yield as compared with the inducible expression. The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm. RNA analysis together with protein measurements show that the PAP gene is poorly transcribed and the PAP mRNA is poorly translated when a lac operator sequence is placed in front of the Shine/Dalgarno (SD) sequence. Nucleotide folding analysis of the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector. These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.

Base Sequence↗

PubMed1997

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit.

A non-Shine-Dalgamo translational initiator is identified in Escherichia coli. The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E. coli cells. The yield of recombinant protein was double that obtained with the consensus Shine-Dalgarno-sequence-(SD)-driven translation. The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box II) which are complementary to nucleotides 1434-1440 and 507-513, respectively, in 16S rRNA. Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16S rRNA during the translation initiation step. Based on these results, we propose that the two widely separated regions 507-513 (part of helical domain 18) and 1434-1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E. coli 30S ribosomal subunit.

Base Sequence↗