Stanka Stoeva

Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli.

The penicillin amidase (PA) from Escherichia coli belongs to a group of proteolytically processed bacterial enzymes. The mechanism of the maturation of the single polypeptide proenzyme has been studied for the PA from E. coli using a slowly processing mutant proenzyme. The mutant proenzyme was constructed by replacing Thr with Gly in the Thr(263)-Ser(264) bond that must be hydrolysed in active PA. The mutant proenzyme was purified by biospecific affinity chromatography using an immobilized monoclonal antibody against PA. The maturation of the free and covalently immobilized purified proenzyme was studied in vitro. For the free proenzyme the same products with PA activity as observed in homogenates of wild-type PA-producing E. coli cells were found to be formed during this process. A kinetic analysis of the possible inter- and intramolecular processes involved in the maturation demonstrated that unambiguous evidence for the existence of intramolecular processes can only be obtained in systems where intermolecular processes are excluded. The Gly(263)-Ser(264) bond was found to be hydrolysed first in the free and immobilized mutant proenzyme, based on determinations of mass spectra, N-terminal sequences and active site concentrations. In the system with immobilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis. Based on the known three-dimensional structure of the PA from E. coli the same maturation mechanism should apply for the wild-type proenzyme.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.

Complete amino acid sequence of the A chain of mistletoe lectin I

The complete amino acid sequence of the A chain of mistletoe lectin I was determined via Edman degradation sequencing of the N‐terminus and tryptic and endoproteinase Asp‐N overlapping fragments, amino acid analysis and MALDIMS. The data obtained show a great homology with the chains of ribosome‐inactivating proteins such as ricin and abrin with 111 (abrin‐a) and 103 (ricin‐D) amino acid residues conserved, respectively. The knowledge of the primary structure of MLA will have a fundamental impact on elucidating the biological function of medically applied mistletoe lectins on a molecular basis.

Structural properties of Rapana thomasiana grosse hemocyanin: Isolation, characterization and N-terminal amino acid sequence of two different dissociation products

1. The native Rapana thomasiana grosse hemocyanin is dissociated under mild conditions and fractionated into two dissociation products, RHSS1 and RHSS2, with an apparent molecular mass of approximately 250 and approximately 450 kDa, respectively. The two species are present in approximately equivalent amounts. SDS-PAGE analysis reveals that the latter component is a dimer of approximately 250 kDa polypeptide chains. 2. The amino acid compositions, as well as some spectroscopic properties of RHSS1, are very similar to those of RHSS2. After dissociation under mild conditions of the native hemocyanin both species preserve their capability of binding reversibly molecular oxygen. 3. RHSS1 and RHSS2 are sequenced directly from the amino-terminus for 15 and 20 steps, respectively. These parts of the two polypeptide chains are highly homologous but with microheterogeneity associated with some positions. They also exhibit high homology with the N-terminal region of subunits or functional domains of other gastropod Hcs.

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH)

The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% beta-sheet and 29% alpha-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c, indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.

Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity.

This report demonstrates that in vitro activation of human cells with the beta-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK- and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and IL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii.

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.

Multidomain Structure of the Rapana thomasiana (Gastropod) Hemocyanin Structural Subunit RHSS1

Abstract The Rapana thomasiana hemocyanin structural subunit RHSS1 is composed of eight functional dioxygen-binding domains. To determine the multidomain structure, the polypeptide chain of RHSS1 was subjected to limited proteolysis with TPCK-trypsin, elastase and other proteinases. Individual functional units and fragments, containing two or three domains, were isolated and characterized. All domains and fragments were N-terminally sequenced and the order of the dioxygen-binding units in the polypeptide chain of RHSS1 was established.

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.