40 results on '"Gutowicz J"'
Search Results
2. Piezoelectric Tuning Fork Mass Sensors as a Novel Tool for Determination of Antibiotic Activity on Pseudomonas Aeruginosa Biofilm
- Author
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Gula, G., primary, Waszczuk, K., additional, Olszak, T., additional, Majewska, J., additional, Gotszalk, T., additional, Drulis-Kawa, Z., additional, and Gutowicz, J., additional
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- 2011
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3. Evaluation of Pseudomonas aeruginosa biofilm formation using piezoelectric tuning forks mass sensors
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Waszczuk, K., primary, Gula, G., additional, Swiatkowski, M., additional, Olszewski, J., additional, Drulis-Kawa, Z., additional, Gutowicz, J., additional, and Gotszalk, T., additional
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- 2010
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4. Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique
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Krystyna Michalak, Gutowicz, J., and Modrzycka, T.
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Spectrometry, Fluorescence ,Protein Conformation ,Muscles ,Liposomes ,Temperature ,Tryptophan ,Animals ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Rabbits ,In Vitro Techniques ,Phosphatidylinositols ,Fluorescent Dyes - Abstract
Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.
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- 1992
5. Up-regulation of key glycolysis proteins in cancer development
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Nowak Nicole, Kulma Anna, and Gutowicz Jan
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glucose transporter 1 ,hexokinase 2 ,phosphoglucose isomerase ,glyceraldehyde 3-phosphate dehydrogenase ,cancer development ,Biology (General) ,QH301-705.5 - Abstract
In rapid proliferating cancer cells, there is a need for fast ATP and lactate production, therefore cancer cells turn off oxidative phosphorylation and turn on the so called "Warburg effect". This regulating the expression of genes involved in glycolysis. According to many studies, glucose transporter 1, which supplies glucose to the cell, is the most abundantly expressed transporter in cancer cells. Hexokinase 2, is one of four hexokinase isoenzymes, is also another highly expressed enzyme in cancer cells and it functions to enhance the glycolytic rate. The up-regulation of these two proteins has been established as an important factor in promoting development and metastasis in many types of cancer. Furthermore, other enzymes involved in glycolysis pathway such as phosphoglucose isomerase and glyceraldehyde 3-phosphate dehydrogenase, exhibit additional functions in promoting tumor growth in a non-glycolytic way. This review demonstrates the pivotal role of GLUT1, HK2, PGI and GAPDH in cancer development. In particular, we look at how the multifunctional proteins, PGI and GAPDH, affect cancer cell survival. We also present various clinical cancer cases in terms of the overexpression of selected proteins, which may be considered as a therapeutic target.
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- 2018
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6. Piezoelectric Tuning Fork Mass Sensors as a Novel Tool for Determination of Antibiotic Activity on Pseudomonas Aeruginosa Biofilm.
- Author
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Gula, G., Waszczuk, K., Olszak, T., Majewska, J., Gotszalk, T., Drulis-Kawa, Z., and Gutowicz, J.
- Abstract
Abstract: In this work an application of quartz tuning forks (QTF) in P. aeruginosa biofilm growth monitoring is presented. QTF-based mass measurement method was examined regarding its applicability in Pseudomonas aeruginosa biofim mass evaluation during antibiotic treatment. Two antibiotics (gentamicin and ciprofloxacin) were tested on their antibacterial activity. Three independent methods were also applied for the experiments: standard colony count of living cells, crystal violet colorimetric method for biofilm monitoring and atomic force microscopy visualization. Using quartz tuning forks the inhibition of Pseudomonas aeruginosa biofilm formation during antibiotic treatment was observed in correlation with other standard tests. These several experiments proved suitability and potential application of the presented technique in biofilm growth measurement for antibacterial compound activity evaluation. [Copyright &y& Elsevier]
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- 2012
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7. Modification of some properties of spherical lipid membranes induced by the association with fructose‐1,6‐bisphosphate aldolase
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Langner, M., Gutowicz, J., and Gomułkiewicz, J.
- Abstract
The influence of fructose‐1,6‐bisphosphate aldolase, as a membrane peripheral protein, on some electrical and transport properties of spherical lipid membranes was investigated. It was found that the association of the enzyme with the membrane did not effect markedly the electrical conductance or capacity of the membrane but decreased the water filtration coefficient and the cationic transferance number. The enzyme association also modifies temperature characteristics of the membrane parameters.
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- 1986
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8. Efflux-mediated antimicrobial multidrug resistance,Oporność wielolekowa zwia̧zana z aktywnym usuwaniem leków z komórek drobnoustrojów
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Jarmuła, A., Ewa Obłąk, Wawrzycka, D., and Gutowicz, J.
9. The association of glycolytic enzymes with cellular and model membranes
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Gutowicz, J. and Grzegorz Terlecki
10. Cysteine peptidase inhibitors and activator(s) in urine of patients with colorectal cancer
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Siewinski, M., Gutowicz, J., Kielan, W., and Bolanowski, M.
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Colorectal cancer -- Diagnosis ,Cysteine -- Physiological aspects ,Business ,Health care industry - Abstract
According to the authors' abstract of an article published in Oncology, 'Th e total activity of cysteine peptidase inhibitors and activator(s) was determined in the samples of urine received from [...]
- Published
- 1994
11. Innate immune properties of selected human neuropeptides against Moraxella catarrhalis and nontypeable Haemophilus influenzae
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Augustyniak Daria, Jankowski Adam, Mackiewicz Paweł, Skowyra Agnieszka, Gutowicz Jan, and Drulis-Kawa Zuzanna
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Neuropeptide Y ,Substance P ,CGRP ,Somatostatin ,Killing ,Permeabilization ,Phagocytosis ,Immunomodulation ,Moraxella catarrhalis ,Haemophilus influenzae ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background Considerable evidence supports the concept of active communication between the nervous and immune systems. One class of such communicators are the neuropeptides (NPs). Recent reports have highlighted the antimicrobial activity of neuropeptides, placing them among the integral components of innate immune defense. This study examined the action of four human neuropeptides: calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and somatostatin (SOM), which are accessible in the upper respiratory tract, against two human-specific respiratory pathogens. We studied: (i) neuropeptide-mediated direct antibacterial activity exerted against Moraxella catarrhalis and nontypeable Haemophilus influenzae, and (ii) indirect immunomodulatory role of these neuropeptides in the neutrophil-mediated phagocytosis of indicated pathogens. Results We found that 100 micromolar concentrations of CGRP, NPY, SP, and SOM effectively permeabilized bacterial membranes and showed (except SOM) bactericidal activity against both pathogens. SOM acted only bacteriostatically. However the killing efficacy was dependent on the bactericidal assay used. The rank order of killing NP effect was: NPY ≥ CGRP > SP >> SOM and correlated with their potency to permeabilize bacterial membranes. The killing and permeabilization activity of the analyzed NPs showed significant correlation with several physicochemical properties and amino acid composition of the neuropeptides. M. catarrhalis was more sensitive to neuropeptides than nontypeable H. influenzae. The immunomodulatory bimodal effect of physiological concentrations of CGRP, NPY, and SP on the phagocytic function of human neutrophils against M. catarrhalis and H. influenzae was observed both in the ingestion (pathogen uptake) and reactive oxygen species generation stages. This effect was also dependent on the distinct type of pathogen recognition (opsonic versus nonopsonic). Conclusions The present results indicate that neuropeptides such as CGRP, NPY, and SP can effectively participate in the direct and indirect elimination of human-specific respiratory pathogens. Because the studied NPs show both direct and indirect modulating antimicrobial potency, they seem to be important molecules involved in the innate host defense against M. catarrhalis and nontypeable H. influenzae.
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- 2012
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12. Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family.
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Kędzior M, Pawlak A, Seredyński R, Bania J, Platt-Samoraj A, Czemplik M, Klausa E, Bugla-Płoskońska G, and Gutowicz J
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- Animals, Cattle, Cysteine Proteases metabolism, DNA-Binding Proteins, Escherichia coli metabolism, Escherichia coli Proteins, Humans, Molecular Chaperones, Papain metabolism, Cathepsins metabolism, Cell Extracts chemistry, Cysteine Proteinase Inhibitors metabolism, Papain antagonists & inhibitors, Yersinia enterocolitica metabolism
- Abstract
Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5-9), and have high molecular weights. Batch affinity chromatography on papain-agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC-MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp., (Copyright © 2017 Elsevier GmbH. All rights reserved.)
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- 2018
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13. Different patterns of extracellular proteolytic activity in W303a and BY4742 Saccharomyces cerevisiae strains.
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Seredyński R, Wolna D, Kędzior M, and Gutowicz J
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- Caseins metabolism, Electrophoresis, Polyacrylamide Gel, Gelatin metabolism, Hydrogen-Ion Concentration, Peptide Hydrolases chemistry, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae metabolism, Substrate Specificity, Peptide Hydrolases metabolism, Proteolysis, Saccharomyces cerevisiae enzymology
- Abstract
Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
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- 2017
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14. Microbial inhibitors of cysteine proteases.
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Kędzior M, Seredyński R, and Gutowicz J
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- Cysteine Proteinase Inhibitors chemistry, Substrate Specificity, Virulence Factors chemistry, Virulence Factors metabolism, Archaea metabolism, Bacteria metabolism, Cysteine Proteases metabolism, Cysteine Proteinase Inhibitors metabolism, Fungi metabolism, Viruses metabolism
- Abstract
Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.
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- 2016
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15. Inhibition of cathepsin B activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
- Author
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Kędzior M, Seredyński R, Godzik U, Tomczyk D, Gutowicz J, Terlecka E, Całkosiński I, and Terlecki G
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- Animals, Cattle, Dioxins, Humans, Cathepsin B antagonists & inhibitors, Polychlorinated Dibenzodioxins toxicity
- Abstract
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent toxic isomer in the dioxin-like family. Due to its resistance to metabolic degradation, this ubiquitous environmental pollutant readily accumulates in multiple organs. Cathepsin B is a lysosomal cysteine protease playing an essential role in the intracellular protein turnover. Alterations in its expression, activity, and localization may facilitate the development of many pathologies, including cancer. TCDD, due to its extremely lipophilic nature, may diffuse through biological membranes and affect lysosomal enzymes, including cathepsins. Therefore, in this study we performed two enzymatic assays, spectrofluorimetry and gelatin zymography, in order to evaluate the effect of TCDD on purified bovine cathepsin B. We showed that the dioxin decreases the enzyme's activity in a dose-dependent manner. The reversibility of TCDD-induced inhibition of the protease was also examined, suggesting that TCDD does not bind covalently to the enzyme's active site, acting rather as a reversible inhibitor.
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- 2015
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16. [Efflux-mediated antimicrobial multidrug resistance].
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Jarmuła A, Obłąk E, Wawrzycka D, and Gutowicz J
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- Bacteria drug effects, Bacterial Proteins, Biological Transport, Active, Fungi drug effects, Humans, Anti-Bacterial Agents pharmacology, Antifungal Agents pharmacology, Drug Resistance, Multiple, Bacterial, Drug Resistance, Multiple, Fungal
- Abstract
Multidrug resistance is a major problem in the treatment of infectious diseases caused by bacteria and fungi. One of the basic mechanisms of resistance is active efflux of distinct drugs from cells. Export of toxic compounds from bacterial cells is mediated by proteins of 5 distinct families: MF, SMR, ABC, RND and MATE. The substrate spectrum of efflux pumps includes antibiotics, chemotherapeutics and detergents. Genes that determine resistance can be located on chromosomes or mobile elements (plasmids, transposons, integrons). The presence of resistance genes on mobile elements enables bacteria to transfer those genes between cells and spread the multidrug resistance phenotype. There are several inhibitors of efflux pumps that are currently in the experimental phase. Proteins that mediate multidrug resistance are also present in fungal cells. They belong mainly to the ABC superfamily of transporters and PDR subfamily. These efflux pumps are widely investigated in Saccharomyces cerevisiae.
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- 2011
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17. The immunogenicity of the liposome-associated outer membrane proteins (OMPs) of Moraxella catarrhalis.
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Augustyniak D, Mleczko J, and Gutowicz J
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- Adolescent, Animals, Antibodies blood, Antibodies metabolism, Bacterial Outer Membrane Proteins isolation & purification, Bacterial Outer Membrane Proteins metabolism, Blotting, Western, Child, Cross Reactions, Enzyme-Linked Immunosorbent Assay, Female, Humans, Immunoglobulin A metabolism, Immunoglobulin G metabolism, Liposomes chemistry, Mice, Mice, Inbred BALB C, Micelles, Moraxella catarrhalis isolation & purification, Phosphatidylcholines chemistry, Bacterial Outer Membrane Proteins immunology, Moraxella catarrhalis immunology
- Abstract
The outer membrane proteins (OMPs) are the most immunogenic and attractive of the Moraxella catarrhalis vaccine antigens that may induce the protective immune response. The aim of this study was to determine the effectiveness of two types of OMP-associated phosphatidylcholine (PC) liposomal formulations (OMPs-PC, PC-OMPs) and of Zwittergent-based proteomicelles (OMPs-Z) in potentiating an anti-OMP systemic immune response in mice. The immunogenicities of the above preparations were evaluated by assessing serum anti-OMP IgG and IgA reactivity in the post-immunized mouse antisera using ELISA and Western blotting. Additionally, the cross-reactivity of the most effective anti-OMP response was determined using heterologous sera from both humans and mice. Both the proteoliposomes and the proteomicelles showed high immunogenic properties and did not elicit any distinct quantitative differences in the antibody titer or qualitative differences in the pattern of the mouse antisera. The post-immunized mouse antisera predominantly recognized a approximately 60-kDa OMP of M. catarrhalis. That protein was also found to be a highly cross-reactive antigen interacting with a panel of pooled mouse antisera produced by immunization either with whole cells or the purified OMPs of heterologous M. catarrhalis strains. Furthermore, normal sera collected from healthy children were found to be preferentially reactive with the 60-kDa OMP. The serum-specific IgG, IgA and IgM were respectively detected via immunoblotting in 90%, 85% and 30% of heterologous human sera. This similar immunogenic effectiveness of both OMP-associated liposomal formulations could contribute to the practical use of such formulations in the future in human vaccination. Moreover, the highly cross-reactive 60-kDa OMP seems to be an important antigenic marker of M. catarrhalis, and, as it is responsible for the induction of an antibody-mediated and long-lasting immune response, studying it may partially aid us in understanding the relatively low degree of pathogenicity of the bacterium in immunocompetent individuals.
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- 2010
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18. Distinct roles of the last transmembrane domain in controlling Arabidopsis K+ channel activity.
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Gajdanowicz P, Garcia-Mata C, Gonzalez W, Morales-Navarro SE, Sharma T, González-Nilo FD, Gutowicz J, Mueller-Roeber B, Blatt MR, and Dreyer I
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- Amino Acid Sequence, Animals, Arabidopsis chemistry, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Computer Simulation, Models, Biological, Models, Molecular, Models, Structural, Mutation, Oocytes, Potassium Channels, Inwardly Rectifying metabolism, Sequence Alignment, Shaker Superfamily of Potassium Channels genetics, Shaker Superfamily of Potassium Channels metabolism, Xenopus, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Cell Membrane metabolism, Potassium Channels, Inwardly Rectifying chemistry, Potassium Channels, Voltage-Gated chemistry, Potassium Channels, Voltage-Gated metabolism, Shaker Superfamily of Potassium Channels chemistry
- Abstract
The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (K(in)) channels that allow plant cells to accumulate K(+) and outward-rectifying (K(out)) channels that mediate K(+) efflux. Despite their close structural similarities, the activity of K(in) channels is largely independent of K(+) and depends only on the transmembrane voltage, whereas that of K(out) channels responds to the membrane voltage and the prevailing extracellular K(+) concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). Here we investigated the functional equivalence of the S6 helices of the K(in) channel KAT1 and the K(out) channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K(+)-sensor from K(out) to K(in) channels.
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- 2009
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19. Inhibitory activity against papain, a CA1 cysteine peptidase, in Saccharomycetaceae.
- Author
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Gawlik K and Gutowicz J
- Subjects
- Culture Media chemistry, Fungal Proteins isolation & purification, Papain metabolism, Protease Inhibitors isolation & purification, Saccharomycetales drug effects, Fungal Proteins pharmacology, Papain antagonists & inhibitors, Protease Inhibitors pharmacology, Saccharomycetales enzymology, Saccharomycetales metabolism
- Abstract
A search for new biological sources of cysteine peptidase inhibitors has not only an academic aspect but is of great importance in medicine and biotechnology. The activity of CA1 peptidases can be inhibited by proteins of nine structurally different families. Although these inhibitors are widespread in nature, there is little information on them in yeast and in the kingdom of fungi overall. To gain insight into the endogenous inhibitors of CA1 cysteine peptidases in unicellular fungi, we initiated a study of the extra- and intracellular antipapain activity in yeast. We report here, for the first time, an analysis of the inhibitory activity against papain in the culture medium and the cell-free extract of 16 yeast strains belonging to the Saccharomycetaceae family. The existence of the antipapain activity, likely from protein inhibitors, in all the tested yeast strains has been demonstrated.
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- 2008
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20. Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH.
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Terlecki G, Czapiñska E, Rogozik K, Lisowski M, and Gutowicz J
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- Animals, Cardiolipins chemistry, Cardiolipins metabolism, Circular Dichroism, Hydrogen-Ion Concentration, In Vitro Techniques, Kinetics, L-Lactate Dehydrogenase chemistry, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Liposomes, Phosphatidylcholines chemistry, Phosphatidylcholines metabolism, Phosphatidylserines chemistry, Phosphatidylserines metabolism, Phospholipids chemistry, Protein Conformation, Substrate Specificity, Swine, L-Lactate Dehydrogenase metabolism, Muscle, Skeletal enzymology, Phospholipids metabolism
- Abstract
Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.
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- 2006
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21. [Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases].
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Gawlik K, Poreba W, and Gutowicz J
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- Animals, Humans, Structure-Activity Relationship, Cystatins pharmacology, Cysteine Endopeptidases drug effects, Protease Inhibitors classification, Protease Inhibitors pharmacology
- Abstract
Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.
- Published
- 2005
22. The association of glycolytic enzymes with cellular and model membranes.
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Gutowicz J and Terlecki G
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- Animals, Cell Membrane metabolism, Humans, Kinetics, Lipid Bilayers metabolism, Protein Binding, Protein Conformation, Cell Membrane enzymology, Glycolysis
- Abstract
This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity
- Published
- 2003
23. Inhibition of cathepsin B activity in human breast cancer tissue by cysteine peptidase inhibitor isolated from human placenta: immunohistochemical and biochemical studies.
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Saleh Y, Siewiński M, Sebzda T, Jeleń M, Ziółkowski P, Gutowicz J, Gryboś M, and Pawelec M
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- Breast cytology, Breast metabolism, Breast Neoplasms pathology, Cathepsin B antagonists & inhibitors, Female, Humans, Placenta chemistry, Breast Neoplasms metabolism, Cathepsin B metabolism, Cysteine Proteinase Inhibitors metabolism, Placenta metabolism
- Abstract
Cysteine peptidases and their endogenous inhibitors (CPI) have been shown to be involved in tumor progression and metastasis. Since their activity has been found to be changed in tumor tissue and/or body fluids of cancer patients, the determination of the peptidase/inhibitor levels is considered as a procedure of diagnostic value. Determination of cathepsin B, its precursor and inhibitor activity in homogenates of tumors and control breast tissue samples of patients with invasive ductal and lobular breast carcinoma and with benign breast disease (BBD) was performed using fluorometric assay. Immunohistochemical staining of the breast tissue samples was carried out using polyclonal antibody against cysteine peptidase inhibitor isolated from human placenta. Procathepsin B and cathepsin B were found to be significantly increased and their endogenous inhibitors decreased in homogenates of tumors from patients with breast cancer. A correlation between procathepsin B or cathepsin B activities as well as cysteine peptidase inhibitor activity and the histopathological grading of the tumor was observed. All samples of the tumor tissue showed positive immunostaining with antibody raised against cysteine peptidase inhibitor, while in the control tissue samples the immunostaining was much weaker. Significant difference observed between the activities of cathepsin B and/or its precursor in malignant and benign tumors might serve as a useful clinical indicator in discrimination between benign and invasive tumors.
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- 2003
24. Activity of cysteine proteases and their inhibitors in the lymph nodes of larynx cancer patients.
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Siewiński M, Berdowska I, Mikulewicz W, Wnukiewicz J, and Gutowicz J
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- Humans, Laryngeal Neoplasms etiology, Lymphatic Metastasis, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors metabolism, Laryngeal Neoplasms metabolism, Lymph Nodes metabolism
- Abstract
Background: Cysteine endopeptidases and their inhibitors play an important role in the process of carcinogenesis. Positive correlation has been found between tumor invasiveness, its metastatic potential and the secretion of cysteine endopeptidases. Cysteine protease inhibitory activity is also altered in malignant tumors and various body fluids of patients with cancer., Material/methods: Total cysteine endopeptidase activity and cysteine proteinase inhibitory activity were measured in homogenates of cervical lymph node tissue surgically obtained from the larynx of cancer patients. The tissue samples were histologically examined, and each was divided into two parts: positive (PCN), with mostly cancer cells, and negative (NCN), with no cancer cells., Results: In the PCNs, the levels of the assayed enzymes and their inhibitors were significantly higher than in the NCNs. The mean values of cysteine protease activity were 2.70I2.29 and 1.59I1.28 for PCNs and NCNs, respectively (p<0.005). The mean values of cysteine protease inhibitors were 9.1I8.6 and 6.1I6.3 for PCNs and NCNs, respectively (p<0.02). An altered protease-inhibitor activity ratio was also found in PCN samples compared to NCNs., Conclusions: These data suggest increased activity of cysteine peptidases and their inhibitors in the case of secondary tumor tissue. The cancer cells metastasized to lymph node tissue produce some alteration in balance between cysteine protease activity and the endogenous inhibitors of the proteases.
- Published
- 2002
25. [Cathepsin B and neoplasm invasiveness].
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Poreba W, Gawlik K, and Gutowicz J
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- Animals, Cathepsin B genetics, Humans, Lysosomes metabolism, Cathepsin B metabolism, Neoplasm Invasiveness physiopathology
- Published
- 2002
26. Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine.
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Terlecki G and Gutowicz J
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- Animals, Glycolysis, Kinetics, Muscle, Skeletal enzymology, Spectrometry, Fluorescence methods, Swine, L-Lactate Dehydrogenase metabolism, Lipid Bilayers, Phosphatidylserines metabolism
- Abstract
Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.
- Published
- 2002
27. The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies.
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Terlecki G, Czapińska E, and Gutowicz J
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- Animals, Glycolysis, Hydrogen-Ion Concentration, Kinetics, Liposomes metabolism, Muscle, Skeletal enzymology, Phospholipids metabolism, Swine, L-Lactate Dehydrogenase chemistry, L-Lactate Dehydrogenase metabolism, Phosphatidylserines chemistry, Phosphatidylserines metabolism
- Abstract
Lactate dehydrogenase is one of the enzymes of the glycolytic path. It has been shown to be able to bind in vitro to cellular membranes. The presence of anionic phospholipids induces changes in the catalytic properties of the enzyme similar to those found when the enzyme is bound to natural membranes. In this study, a nonionic detergent (Tween 20), at concentrations not affecting the catalytic activity of LDH, was used to study the role of the lipid supra-molecular structure in the interaction between pig skeletal muscle lactate dehydrogenase and phosphatidylserine. Tween 20 changes the equilibrium of concentrations between the lipid supra-molecular forms. The detergent at the used concentration values did not alter the activity of the enzyme when it was used on its own, but did diminish the level of inhibition induced by the studied phospholipid. The obtained results showed that the interaction is reversible and that the bilayer structure of the lipid is essential for the inhibition.
- Published
- 2002
28. Role of cysteine endopeptidases in cancerogenesis.
- Author
-
Siewinski M, Gutowicz J, Zarzycki A, and Mikulewicz W
- Subjects
- Cell Transformation, Neoplastic, Humans, Neoplasm Invasiveness, Neoplasm Metastasis, Neoplasms enzymology, Cysteine Endopeptidases physiology, Neoplasms etiology
- Abstract
Role of cysteine endopeptidases in cancerogenesis steps: neoplastic transformation, invasion and metastasis is reviewed and discussed. Positive correlation between tumor invasiveness, as well as its metastatic potential and secretion of cysteine endopeptidase (particularly cathepsins B and L) has been documented well in literature. Based on our recent results we postulate that serum endopeptidase-like activity could be used as a marker of cancer aggressiveness in diagnostic procedures in oncology. We also propose that the cysteine endopeptidase inhibitor levels (total, active and latent) could be useful factors for recognising the activation of the organism self-defence mechanisms against cancer. In addition, our idea of use of urinary cysteine peptidase inhibitors (UCPI) as potential anticancer agents is presented and discussed.
- Published
- 1996
- Full Text
- View/download PDF
29. Interaction of bovine heart pyruvate kinase with phospholipids.
- Author
-
Dabrowska A, Terlecki G, Czapińska E, and Gutowicz J
- Subjects
- Animals, Binding Sites, Cattle, Enzyme Activation drug effects, Kinetics, Liposomes, Magnesium Chloride pharmacology, Phosphatidylserines pharmacology, Membrane Lipids pharmacology, Myocardium enzymology, Phospholipids pharmacology, Pyruvate Kinase metabolism
- Abstract
The interaction between bovine heart pyruvate kinase and liposomes was investigated for various phospholipids as function of pH, and salt concentration using steady-state kinetics and ultracentrifugation. Liposomes made from erythrocyte total lipid fraction and individual phospholipids were used. Pyruvate kinase specific activity increases upon the interaction with the phospholipids. The activation is specifically sensitive to presence of phosphatidylserine in liposomes. L-serine, and phospho-L-serine which are main components of phosphatidylserine head group show also some activation effect. Efficient adsorption of pyruvate kinase to phosphatidylserine liposomes occurs in the pH range 6.0-8.0 and at low ionic strength. Interaction with phosphatidylserine liposomes results in the change of Vmax and Km values for phospho enol pyruvate without marked effect on Km value for ADP, and Hill coefficients for both substrates. The interaction does not seem to influence the cooperativity between binding sites.
- Published
- 1995
- Full Text
- View/download PDF
30. Cysteine peptidase inhibitors and activator(s) in urine of patients with colorectal cancer.
- Author
-
Siewiński M, Gutowicz J, Kielan W, and Bolanowski M
- Subjects
- Adult, Aged, Analysis of Variance, Female, Humans, Male, Middle Aged, Papain antagonists & inhibitors, Colorectal Neoplasms urine, Cysteine Proteinase Inhibitors urine
- Abstract
The total activity of cysteine peptidase inhibitors and activator(s) was determined in the samples of urine received from colorectal cancer patients. Patients with peptic ulcer and healthy volunteers agreed to be a control group. The studies revealed a marked difference between the values of the determined parameters for the patients with colorectal cancer and those for the control group. Determination of cysteine peptidase inhibitors in patient's urine is proposed as a new diagnostic procedure.
- Published
- 1994
- Full Text
- View/download PDF
31. Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique.
- Author
-
Michalak K, Gutowicz J, and Modrzycka T
- Subjects
- Animals, Fluorescent Dyes, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, In Vitro Techniques, Muscles enzymology, Phosphatidylinositols, Protein Conformation, Rabbits, Spectrometry, Fluorescence, Temperature, Tryptophan chemistry, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Liposomes
- Abstract
Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.
- Published
- 1992
32. Adsorption of bovine muscle lactate dehydrogenase to erythrocyte membranes.
- Author
-
Dabrowska A, Gutowicz J, and Terlecki G
- Subjects
- Adsorption, Animals, Cattle, Humans, Hydrogen-Ion Concentration, In Vitro Techniques, Liposomes, Muscles enzymology, NAD metabolism, Erythrocyte Membrane metabolism, L-Lactate Dehydrogenase metabolism
- Published
- 1990
33. Fluorescent probe studies on binding of glyceraldehyde-3-phosphate dehydrogenase to phosphatidylinositol liposomes. Further evidence for conformational changes.
- Author
-
Michalak K, Gutowicz J, and Modrzycka T
- Subjects
- Animals, Binding Sites, Energy Transfer, Muscles enzymology, Protein Binding, Protein Conformation, Rabbits, Spectrometry, Fluorescence, Urea, Glycerolphosphate Dehydrogenase metabolism, Liposomes metabolism, Phosphatidylinositols metabolism
- Abstract
Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle can be absorbed on charged lipid bilayers by electrostatic forces. Upon binding to phosphatidylinositol liposomes the enzyme modifies its conformational state as it is shown by resonance energy transfer experiments. In the presence of 2-mercaptoethanol o-phthaldialdehyde reacts with amino groups of the protein and the covalently bound fluorophore is an acceptor of excitation energy transferred from tryptophanyl residues of the protein. The observed decrease of energy transfer efficiency upon binding to phosphatidylinositol liposomes is compared with the influence of the urea on the fluorescence spectra of the labelled protein. Significance of conformational changes of the enzyme upon adsorption on liposomes in the regulating function of cell membranes is discussed.
- Published
- 1987
- Full Text
- View/download PDF
34. Interaction of bovine heart lactate dehydrogenase with erythrocyte lipids.
- Author
-
Dabrowska A and Gutowicz J
- Subjects
- Adsorption, Animals, Cattle, Hydrogen-Ion Concentration, Liposomes metabolism, NAD metabolism, Osmolar Concentration, Phospholipids metabolism, Ultracentrifugation, Erythrocytes metabolism, L-Lactate Dehydrogenase blood, Lipids blood, Myocardium enzymology
- Abstract
The interaction between bovine heart lactate dehydrogenase and erythrocyte lipid suspension as a function of pH, NAD, NADH, lipid and salt concentration was studied by ultracentrifugation. In the presence of erythrocyte lipid liposomes the enzyme forms two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. The two complexes reveal different dependence of their stability on pH values. Lactate dehydrogenase decreases its specific activity when it binds to the phospholipid molecules. Efficient adsorption of lactate dehydrogenase to liposomes occurs in their pH range 6.0-8.0 and at low ionic strength. The adsorption is diminished in the presence of NAD+ but it is not influenced by NADH. Possible mechanisms of the interaction and implications for the function in vivo are discussed.
- Published
- 1986
- Full Text
- View/download PDF
35. Interaction of rabbit muscle aldolase with phospholipid liposomes.
- Author
-
Gutowicz J and Modrzycka T
- Subjects
- Animals, Kinetics, Muscles enzymology, Osmolar Concentration, Protein Conformation, Rabbits, Spectrometry, Fluorescence, Tryptophan, Fructose-Bisphosphate Aldolase metabolism, Liposomes, Phospholipids
- Abstract
The interaction between rabbit muscle fructose diphosphate aldolase and phospholipid model membranes (liposomes) was studied by measurement of the tryptophan fluorescence of the enzyme. Interaction with liposomes decreases intrinsic fluorescence intensity of the enzyme and shifts the emission wavelength maximum to higher values. The effects appear to be strongly dependent on the nature of the phospholipid polar group and on ionic strength. Also, a reversible modification of specific activity of aldolase upon interaction with liposomes was found. It is postulated that aldolase binds to liposomes mainly by electrostatic interactions and that the binding causes a change in the conformation of the enzyme.
- Published
- 1979
- Full Text
- View/download PDF
36. Fluorescence investigation on conformational state of rabbit muscle aldolase interacting with phosphatidylinositol liposomes.
- Author
-
Gutowicz J and Kosmider-Schmidt A
- Subjects
- Animals, Kinetics, Protein Conformation, Rabbits, Spectrometry, Fluorescence, Fructose-Bisphosphate Aldolase metabolism, Liposomes, Muscles enzymology, Phosphatidylinositols metabolism
- Abstract
Evidence of conformational changes in rabbit muscle aldolase upon binding to phosphatidylinositol liposomes and the effect of the interaction on the thermal conformational transition are reported. Interaction with phosphatidylinositol liposomes significantly decreases the aldolase tryptophanyl fluorescence and shifts the maximum wavelength to higher values. The dynamic quenching constant for the aldolase fluorescence quenching by acrylamide in the presence of liposomes is much higher than that for unmodified enzyme; this signifies an increase in accessibility of some tryptophanyl residues to small polar molecules. Indirect interaction between single phospholipid molecules, small micelles or any soluble impurities able to penetrate into the protein molecule interior does not seem to be involved in the conformational rearrangement. Native and liposome-interaction-induced conformational states reveal different temperature dependences of the tryptophan residues exposure. The implications of the modification of the conformational state of the enzyme for its function in vivo are discussed.
- Published
- 1987
- Full Text
- View/download PDF
37. Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme.
- Author
-
Dabrowska A, Terlecki G, and Gutowicz J
- Subjects
- Animals, Binding Sites, Cattle, Cell-Free System, Hydrogen-Ion Concentration, Isoenzymes, Liposomes, Ultracentrifugation, L-Lactate Dehydrogenase metabolism, Muscles enzymology, Myocardium enzymology
- Abstract
The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H4 from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M4, and NADH favors the dissociation.
- Published
- 1989
- Full Text
- View/download PDF
38. Effect of 1-anilinonaphthalene-8-sulphonate on phase transition temperature of dipalmitoylphosphatidylcholine liposomes.
- Author
-
Gutowicz J and Krawczyk A
- Subjects
- Calorimetry, Differential Scanning, Hydrogen-Ion Concentration, Models, Biological, Osmolar Concentration, Anilino Naphthalenesulfonates, Fluorescent Dyes, Lipid Bilayers, Pulmonary Surfactants
- Abstract
The effect of 1-anilinonaphthalene-8-sulfonate (ANS) on the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by differential scanning calorimetry. The main phase transition temperature was found to be shifted to lower values in the presence of the probe. The shift strongly depends on pH and the presence of salts. This indicates that the penetration of the probe of the hydrocarbon moiety of the bilayer is influenced by coulombic interactions. Pretransition phenomena are also affected. The implications for the interpretation of experimental data of biomembrane studies are discussed.
- Published
- 1986
- Full Text
- View/download PDF
39. Binding of glyceraldehyde-3-phosphate dehydrogenase to phospholipid liposomes.
- Author
-
Gutowicz J and Modrzycka T
- Subjects
- Animals, Kinetics, Muscles enzymology, Protein Binding, Rabbits, Glyceraldehyde-3-Phosphate Dehydrogenases, Liposomes, Phospholipids
- Abstract
The binding of glyceraldehyde-3-phosphate dehydrogenase prepared from rabbit muscle to phospholipid model membranes (liposomes) as a function of pH, ionic strength, and the influence of the binding on specific activity of the enzyme was studied. The binding decreases the specific activity of the enzyme. The binding was studied by the method of association of the enzyme with liposomes during centrifugation. The existence of a dominant interaction of electrostatic character was found.
- Published
- 1978
- Full Text
- View/download PDF
40. Liposome-interaction induced conformation changes of glyceraldehyde-3-phosphate dehydrogenase.
- Author
-
Gutowicz J and Modrzycka T
- Subjects
- Phospholipids, Protein Binding, Protein Conformation, Protein Denaturation, Spectrometry, Fluorescence, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Liposomes metabolism
- Abstract
Tryptophanyl emission spectra of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were measured after the addition of liposomes prepared of natural phospholipids: phosphatidylinositols (PI), phosphatidylserines (PS) and phosphatidylcholines (PC). The measurings were made for various molar lipid/protein ratios (100-1000). A decrease in the enzyme fluorescence intensity and a "red" shift of the emission band maximum were observed. The susceptibility of the enzyme fluorescence to liposome action strongly depended on the kind of phospholipid and changed in the sequence PI greater than PS greater than PC. The presence of liposomes affected the accessibility of tryptophan residues for the fluorescence quencher (acrylamide). The results suggested that interaction induces some specific conformation changes in the enzyme molecules which may be responsible for modification of the enzyme activity. A comparison of the modification in fluorescence characteristics with those observed during denaturation suggested that the denaturation mechanism is not operative. Other possible mechanisms of the interaction are discussed.
- Published
- 1986
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