From estrogens via alkaloids to enzymes

5 downloads 0 Views 482KB Size Report
Hugo Theorell, the Nobel Prize laureate for physiol- ogy and medicine in 1955, who offered me a one-year. Unauthenticated. Download Date | 9/24/15 11:35 PM ...
Biologia, Bratislava, 61/3: 333—338, 2006 Section Cellular and Molecular Biology DOI: 10.2478/s11756-006-0061-5

Recollections

From estrogens via alkaloids to enzymes Oskar Markovič Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia; e-mail: [email protected]

Abstract: This personal and professional autobiography covers the 57-year period from 1948 to 2005 and includes the studies at the Faculty of Chemical Technology of the Slovak Technical University and the Faculty of Pharmacy of Comenius University, both in Bratislava, as well as the activities at the Regional Pharmaceutical Research Institute and the Institute of Chemistry of the Slovak Academy of Sciences in Bratislava, the Institute of Organic Chemistry and Biochemistry in Prague, Karolinska Institutet in Stockholm and at other research and scientific working places in Slovakia and abroad. It highlights the research on natural substances and enzymes, especially the pectin methyl esterase.

I was born on January 14, 1927 in Veľké Ripňany, a village in the middle Slovakia, in a teacher’s family. The high-school studies I absolved in Public Gymnasium in Slovak historical town Nitra during 1938–1946 (I started and finished the school in the Czecho-Slovak Republic; from 1939 to 1945 there was the Slovak Republic). In 1946 I started the study at the Faculty of Chemical Technology of the Slovak Technical University in Bratislava with the specialization Organic technology – pharmaceuticals. In 1948 I spent a stay at the Department of Biological Control that belonged to the pharmaceutical factory Slovakofarma, Hlohovec, Slovakia. MUDr. F. V. Selecký was the head of that Department and Dipl. Eng. L. Molnár was a person responsible for the chemical laboratory. The work was focused mainly on the study of estrogenic hormones and control of the products, especially the biological titration of estron. The development of pharmaceutical industry and research in Slovakia was presented in a Slovak pharmaceutical magazine (Molnár & Záthurecký, 1994). After finishing the Slovak Technical University I started to work in the Department of Pharmacology of the newly established Regional Pharmaceutical Research Institute in Bratislava (Fig. 1). The head of that Institute was Dr. F. Selecký. Here I continued the work on the project of estrogenes, mainly on chemical and biological estimation of estron and on preparation of active derivatives of estron (β-estradiol, estradiol benzoate and estradiol dipropionate), as well as their crystal suspensions for intramuscular application (Markovič et al., 1955). In 1955 I was awarded the Prize of Slovak Academy of Sciences after finishing the project entitled “Research of esterogenic substances and preparation of estradiol derivatives”. During 1950-

Fig. 1. The workplace of the Pharmaceutical Research Institute, Bratislava, Mlynské Nivy 37. This building was initially a mill, later it belonged to Medichemia Company and in 1950 it was reconstructed for laboratory purposes.

1954 as the employee of the Pharmaceutical Research Institute I accomplished the study at the Faculty of Pharmacy of the Comenius University in Bratislava. During the study at the Slovak Technical University, there were the lectures on mathematics presented by Prof. Š. Schwarz who achieved an enormous interest of all students. Prof. Schwarz was able to evoke a strong interest in mathematics and he was one of the best pedagogues I knew. The other extraordinary personalities were Prof. Dr. F. Valentin, who gave the lectures in organic chemistry, and professor of pathological anatomy, MUDr. F. Klein, at the Faculty of Pharmacy. These three professors were in their scientific fields outstanding persons, who possessed the genial ability to simplify

Unauthenticated

c 2006 Institute of Molecular Biology, Slovak Download  AcademyDate of Sciences | 9/24/15 11:35 PM

334

O. Markovič

Fig. 2. The marriage of O. Karvašová with O. Markovič in 1954. The witnesses: O. Chodáková and F. Selecký.

and explain even the most complicated scientific information in an attractive and clear mode. Prof. Schwarz was able to teach the infinitesimal calculus also the students with a lack of “mathematic genes”. In that time (1946) the education of organic chemistry started on a basis of electron theory and in a discussion concerning the possible new system in writing the organic substances formulas Prof. Valentin pronounced the interesting sentence: “I will not be here for a long time, but the carbon atom will still have attached four commas!” Prof. Klein, for example, explained the mechanism of blood coagulation in such a way that the pharmacists yet in 2005 were able to reproduce that mechanism (it was verified at the alumni 50 anniversary meeting). I met Prof. Klein later, after his forced departure from the heading post, as he asked me for some information concerning the crystal suspensions of estradiol benzoate for test application at carcinoma of prostate gland in Banská Bystrica Hospital. In 1954 I did the most important step of my life: I got married with Oľga Karvašová (Fig. 2). The marriage witnesses were Mrs. Oľga Chodáková and MUDr. František Selecký, the latter being my scientific ideal, cordial friend and later godfather of our first daughter. In the Pharmaceutical Research Institute I worked also on alkaloids, especially on derivatives of morphium, which were in that time the products of the Slovakofarma Hlohovec Factory. By use of paper chromatography we succeeded to solve the problem of color change combined with obsolence of morphium injection preparations by separation and identification of morhine oxidation products (Markovič et al., 1954). Later we solved the analytical problem of the injection department of Slovakofarma in Martin, Slovakia, by separation and estimation the content of three components (scopolamine, morphine and ethylmorphine) in the preparation Modiscop (Markovič & Rexová, 1957). During that time period (1952-1957) I was a propagator of paper chromatography methods and electrophoresis and together with Dr. Hána from the Institute of Virology of the Slovak Academy of Sciences, we organized special courses and seminars about these

methods under the patronage of the Slovak Chemical Society. I was involved in the chemical part of the project “Pharmacological research of Gallega off. components”. From the leaves of that plant we prepared the extract Galegol containing the active substance lactogogum and from the seeds we isolated the crystalline alkaloid galegin (Markovič & Dittertová, 1955). From 1953, when the Research Institute of Pharmacy and Biochemistry was attached to the Slovak Academy of Sciences I was entrusted with the working on the project “Research of the bee venom” and after solving the problem of the isolation of bee venom and preparation of the bee venom injection and ointment (Virapin), I elaborated the methods for the estimation and control of the bee venom products. The isolation and chemical characterization of the bee venom components became also the subject of my academic PhD-dissertation (in that time it was a scientific degree “CSc.” - Candidate of Science). I performed this work in the years 1954– 1958. The experimental part of my PhD-thesis I accomplished at the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences in Prague. My working stays in that Institute in 1957 and later in 1968 directed my further professional career towards the study of protein structure and function (Markovič, 2004). In the years 1956–1958 I was accredited by the Dean’s office of the Faculty of Natural Sciences, Comenius University in Bratislava, to present lectures on “Protein Structure” and “Methods in Biochemistry” at the Department of Biochemistry. Exactly in the time of the finishing my PhD-thesis in 1958, the political audits took place in Czechoslovakia in all centrally directed institutions and even in the Slovak Academy of Sciences. The audits were directed by Decree No. 265 of the Government of the Czechoslovak Socialist Republic (Prime minister V. Široký): “Policy of evaluation of class and political reliability” from March 14, 1958 (Šubert, 1992; Karvaš, 2002). Based on the decision of the Presidium of the Slovak Academy of Sciences and a special screen commission I was released from the staff of the Institute of Chemistry due to the supposed conspiracy against the state of my father in law Prof. Imrich Karvaš and my father Oto Markovič more than fifteen years ago. Actual June 30, 1958, also the possibility to defend my PhD-thesis was stopped. After my forced departure from the Slovak Academy of Sciences I started to work in the Research Group of the Diary Industry in Bratislava at the division of control of milk products and in the operation of the newly installed cleaning station of waste water from the diary factory in Galanta. In 1960 the Research Group of Diary Industry was transferred to Žilina, Slovakia, and I obtained the proposal from the Research Institute of Rheumatic Diseases in Piešťany, Slovakia, to work in the biochemical laboratory. Here I was engaged in the pathophysiology of synovial fluid as well

Unauthenticated Download Date | 9/24/15 11:35 PM

Recollections: From estrogens via alkaloids to enzymes

Fig. 3. Some members of the biochemical and clinical departments of the Institute of Rheumatic Diseases in Piešťany, Slovakia, in 1964. In upper row: Dr. D. Žitňan, Dr. K. Trnavský, Dr. S. H¨ uttl and O. Markovič. Dr. E. Kresánek, the head of the clinical laboratory, is in the middle.

Fig. 4. O. Markovič and Ľ. Rexová-Benková with Prof. J. F. Thibault, INRA, Nantes, France, during his visit in the Institute of Chemistry of the Slovak Academy of Sciences in 1980.

as in elaborating the methods for estimation sialic acid, glycoproteins, aminotripeptidase and lacticodehydrogenase in human body fluids at rheumatic fever and other rheumatic diseases. Later I was involved in the project “The ochronotic arthropathy” headed by Prof. Štefan Siťaj, director of the Research Institute of Rheumatic Diseases. This work was awarded the Prize of the Scientific Board of the Czechoslovak Department of Health in 1964. Further I joined the investigations on the pathophysiology of the synovial fluid headed by Dr. Svatopluk H¨ uttl (Fig. 3), and this work was awarded in 1966 a prize from the CIBA Company, Switzerland. During my stay in this Institute I became author or coauthor of 16 papers published in Czechoslovak and foreign medical and biochemical journals. In the year 1964 after the intervention of Prof.

335 F. Šorm, the President of the Czechoslovak Academy of Sciences, I got the chance to present my PhDthesis from 1958 in the Institute of Organic Chemistry and Biochemistry in Prague, what I successfully defended. The results of my PhD-thesis were subjects of six papers. The most important result was the isolation of low-molecular bee venom peptides containing N-terminal histamine (Markovič & Rexová, 1963; Rexová & Markovič, 1963); this finding was later confirmed by isolation and synthesis of histamine containing bee venom peptides by American authors (Nelson & O’Connor, 1968). Based on my request to investigate again and revise the results of card actions from the year 1958, I got the statement from the Department of Education and Science of the Central Committee of the Slovak Communist Party from October 1965, No. 567/65-IG/Má, that in the future I will be allowed to work in centrally directed institutes in a position corresponding to my ability and working results. In 1966 a competition for biochemist was announced at the Institute of Chemistry of the Slovak Academy of Sciences and based on my application I, after eight years, was accepted in the department of pectic substances headed by Assoc. Prof. R. Kohn. The general scope of the Institute was directed towards the chemistry and biochemistry of saccharides, my previous laboratory, headed by Dr. Ľ. Rexová-Benková (Fig. 4), being focused on pectic enyzmes. Ľ. Rexová was concerned with fungal polygalacturonases and I started to work on plant pectinesterases and polygalacturonases. After ten-year work on this project we published together the first comprehensive review on pectic enzymes (Rexová-Benková & Markovič, 1976). Pectic enzymes was the main subject of my research from 1966 until 2004 with some breaks caused by working stays in foreign laboratories. In 1967 I visited again the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences in Prague where I worked on cathepsine D (Keilová et al., 1969). In 1968, after political loosening in Czechoslovakia, I succeeded to travel officially for the first time to West by support of Pharmacia Co., Uppsala, Sweden, to attend the Course of Modern Biochemical Methods at the Uppsala University, led by Prof. Stellan Hjertén. After a one-week stay in Uppsala, the participants of the course got the opportunity to visit some scientific and university institutes in Stockholm. At the Karolinska Institutet I was introduced to Prof. Viktor Mutt, one of the nicest persons I met in my life. I admit that his willingness to help me to establish the contacts with some Swedish scientists was motivated also by the occupation of Czechoslovakia in 1968, because he came from Estonia, a country which suffered similar fate as my motherland. Prof. Mutt recommended me to Prof. Hugo Theorell, the Nobel Prize laureate for physiology and medicine in 1955, who offered me a one-year

Unauthenticated Download Date | 9/24/15 11:35 PM

336

O. Markovič

Fig. 5. Prof. H. Theorell with his wife and O. Markovič during the FEBS Congress in Varna, Bulgaria, 1971.

Fig. 7. R. Kohn, E. Machová, O. Markovič and A. Slezárik - a research group awarded by the Prize of the Slovak Academy of Sciences in 1987.

Fig. 6. Prof. Hans J¨ ornvall in his laboratory with coworkers.

stay at the Biochemical Nobel Institute in Karolinska Institutet, Stockholm (Fig. 5). In 1969 I acceded as a visiting scientist at this prestigious European biochemical institute, where my work was focused on rat liver alcohol dehydrogenase, at that time still not isolated labile enzyme. I succeeded to isolate and characterize this enzyme (Markovič et al. 1971; Markovič & Theorell, 1972) and with Hans J¨ ornvall performed the structural studies of this alcohol dehydrogenase ¨ rnvall & Markovič, 1972). (Jo The cooperation with Prof. Hans J¨ ornvall, who became the head of the Department of Medical Biochemistry and Biophysics on Karolinska Institutet, continued also in the research focused on pectinesterases. The substantial part of structural studies was performed with him and in his coworkers in Karolinska Institutet during my several long- and short-time stays. The work on this common project resulted in ten publications. Without the personal involvement and help of Hans J¨ ornvall (Fig. 6) as well as the grant support from the Swedish Medical Research and Swedish Research Council, it would be impossible to realize the sequence-structural studies of plant pectinesterases. For the active cooperation with the Institute of Chemistry of the Slovak Academy of Sciences, Prof. H. J¨ornvall was awarded by the Slovak Academy of Sciences with

the Memory Medal for merits on Slovak science in 1998. As a result of my work in the Institute of Chemistry I, together with my coworkers, published twentyfour papers on pectinesterases from 1969 up to 1987 and in 1987 my team with Assoc. Prof. R. Kohn, Dr. E. Machová and Ing. A. Slezárik (Fig. 7) was awarded the Prize of Slovak Academy of Sciences for the research project entitled “Pectinesterase - multiplicity and structure-functional relations”. During the studies on pectinesterases (until to year 2002) we achieved the following main results: (i) testing the action of serine inhibitors on pectinesterase enabled us to exclude the assumed hypothesis that this enzyme belongs to the group of serine-type esterases (Markovič & Patočka, 1977); (ii) using the gel electrophoresis and specific detection it was possible to reveal the presence of multiple forms of pectinesterase from tomato fruit for the first time (Markovič, 1974; Rexová-Benková & Markovič, 1976); (iii) in the same pectiesterase the isoleucine as the N-terminal amino acid residue was determined (Markovič & Sajgó, 1977) and a tyrosine was confirmed to play a functional role (Markovič, 1983); (iv) methylesters of oligogalacturonic acids were prepared with predicted degree of esterification, and these were used as substrates for the estimation of the chain length at which the action of plant and fungal pectinesterases starts (Markovič et al. 1981, 1983); (v) we also found that different deestrification mode of pectin with pectineterases does not depend on the enzyme origin but is determined by its character defined by pH-optimum and isoelectric point (Markovič & Kohn, 1984); (vi) with Prof. H. J¨ ornvall we described the primary structures of tomato and carrot pectinesterases ¨ rnvall, 1986, 1990; Markovič et (Markovič & Jo al., 2002) and in tomato and Aspergillus pectinesterases

Unauthenticated Download Date | 9/24/15 11:35 PM

Recollections: From estrogens via alkaloids to enzymes

337

we evaluated the role of histidine residues, which do not have the active-site functions, but contribute to the ¨ rnvall, overall structural stability (Markovič & Jo 1990); (vii) with Prof. Hans Eklund from the Department of Molecular Biology of the Swedish University of Agricultural Sciences in Uppsala, Sweden, we determined the first crystal structure of plant pectinesterase from carrot (Johansson et al. 2002). In 1986 after three years of political-administrative restrictions I got the permission to defend my doctoral dissertation and received from the Presidium of the Czechoslovak Academy of Sciences in Prague the academic degree “DrSc.” in biochemistry. The cooperation with my colleagues from the Institute of Chemistry was also very interesting and fruitful, especially with Peter Biely, Danica Mislovičová and Kvetoslava Heinrichová. It concerned the preparation and application of chromogenic substrates for identification and estimation of cellulases, amylases and polyglacturonases (Biely et al. 1985, 1988; Biely & Markovič, 1988; Markovič et al., 1992). I was also the member of the Dr. Biely’s team, which was awarded the Prize of the Slovak Academy of Sciences in 1997. In 1994 I spent a one-year stay at the Cornell University, Ithaca, NY, USA, in the Department of Seed Biology where I worked with Prof. R. Obendorf on isolation and characterization of soybean pectinesterase (Markovič & Obendorf, 1988). From 1996 as a retiree I continued in the Institute of Chemistry to work with Dr. Eva Stratilová on fungal polygalacturonases focused on the role of tyrosine residue and the glycoprotein character of these enzymes (Stratilová et al. 1996, 1998a) as well as on exopolygalacturonase, pectinesterase and pectin acetylesterase from carrot (Stratilová et al., 1998b). In cooperation with Dr. Štefan Janeček from the Institute of Molecular Biology of the Slovak Academy of Sciences we published two papers on sequence-structural features and phylogenetic relationships of pectin degrading glycoside hydrolases of family 28 (Markovič & Janeček, 2001) and pectin methylesterases (Markovič & Janeček, 2004). The Organizing Committee of the international Symposium on Pectins and Pectinases held in Rotterdam, The Netherlands, in 2001, designated my oral talk on polygalacturonases as an outstanding contribution by Certificate of Merit (Fig. 8). It was rather troublesome for me and I was not very agreeable to write this “self-promotion”, but I am one of the oldest and still living persons within the Slovak Academy of Sciences, who had to undergo the suffering from the communist system in 1958. Up to now there was not performed any historical evaluation of the “events” occurred at that time in the centrally directed institutions. Perhaps this Recollection could show a less known facet of that period of our history of science and point out especially for young generation that the

Fig. 8. Certificate of Merit received by O. Markovič in Pectin and Pectinases Symposium held in Rotterdam in 2001.

Fig. 9: O. Markovič with grand daughters in Low Tatras, Slovakia, in 2004.

fight for truth was really difficult and sometimes also hopeless. Today I am aware that many tragic events related to the decease of persons close to me as well as the struggle for the employment and living standard problems, caused by political invasion of authoritative system, is possible to overcome. It was the support of my family, my friends, sport activities and working results

Unauthenticated Download Date | 9/24/15 11:35 PM

338

O. Markovič

that helped me to overcome all these problems (Fig. 9). I identify myself with the notion of Prof. Žucha who wrote (Hulín & Žucha, 2005): “It is the ability to bear the strokes of fate and proper imperfections that should be the attribute of each person who successfully compete in his/her life.”. References BIELY, P. & MARKOVIČ, O. 1988. Resolution of glycanases of Trichoderma reesei with respect to cellulose and xylan degradation. Biotechnol. Appl. Biochem. 10: 99–106. ´ OVÁ, D. 1985. Sensitive BIELY, P., MARKOVIČ, O. & MISLOVIC detection of endo-1,4-β-glucanases and endo-1,4-β-xylanases in gels. Anal. Biochem. 144: 147–151. ´ OVÁ, D., MARKOVIČ, O. & KALÁČ, V. 1988. BIELY, P., MISLOVIC A new chromogenic substrate for assay and detection of αamylase. Anal. Biochem. 172: 176–179. HULÍN, I. & ŽUCHA, I. 2005. Rozhovory. Faber, Bratislava, p. 14. ¨ RNVALL, JOHANSSON, K., EL-AHMAD, M., FRIEMANN, R., JO H., MARKOVIČ, O. & EKLUND, H. 2002. Crystal structure of plant pectin methylesterase. FEBS Lett. 514: 243–249. ¨ RNVALL, H. & MARKOVIČ, O. 1972. Structural studies of alcoJO hol dehydrogenase from rat liver. Eur. J. Biochem. 29: 167– 174. KARVAŠ, M. 2001. Môj otec Imrich Karvaš. Rak, Budmerice, pp. 96–98. KEILOVÁ, H., MARKOVIČ, O. & KEIL, B. 1969. Characterization and chemical modification of cathepsin D from bovine spleen. Coll. Czech. Chem. Commun. 34: 2154–2157. MARKOVIČ, O. 1974. Tomato pectinesterase – characterization of one of its multiple forms. Coll. Czech. Chem. Commun. 39: 908–913. MARKOVIČ, O. 1978. Pectinesterase from carrot (Daucus carota L.). Experientia 34: 561–562. MARKOVIČ, O. 1983. Modification of tyrosine residues of tomato pectinesterase. Coll. Czech. Chem. Commun.48: 790–797. MARKOVIČ, O. 2004. Čo všetko nám vzalo dvadsať normalizačných rokov. Mosty (Česko-Slovenský dvojtýždenník) 3 AUG 2004, 13: p. 14. MARKOVIČ, O., CEDERLUND, E., GRIFFITHS, W.J., LIPKA, T. ¨ RNVALL, H. 2002. Characterization of carrot pectin & JO methylesterase. Cell. Mol. Life Sci. 59: 513–518. MARKOVIČ, O. & DITTERTOVÁ, V. 1955. Príspevok k izolácii galegínu z rastliny Gallega officinalis. Chem. Zvesti 9: 576– 579. MARKOVIČ, O. & JANEČEK, Š. 2001. Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution. Protein Eng. 14: 615–631. MARKOVIČ, O. & JANEČEK, Š. 2004. Pectin methylesterases: sequence-structural features and phylogenetic relationships. Carbohydr. Res. 339: 2281–2295. ¨ RNVALL, H. 1986. Pectinesterase. The priMARKOVIČ, O. & JO mary structure of the tomato enzyme. Eur. J. Biochem. 158: 455–462. ¨ RNVALL, H. 1990. Tomato and Aspergillus MARKOVIČ, O. & JO niger pectinesterases. Correlation of differences in existing reports: large species variations. Protein Sequence Data Anal. 3: 513–515. MARKOVIČ, O. & KOHN, R. 1984. Mode of pectin deesterification by Trichoderma reesei pectinesterase. Experientia 40: 842– 843.

MARKOVIČ, O., KOVÁČ, P. & SLEZÁRIK, A. 1981. Synthesis and reactions of uronic acid derivatives. XXI. Controlled esterification of oligo-D-galactosiduronic acids with diazomethane. Chem. Zvesti 35: 79–83. MARKOVIČ, O., MACHOVÁ, E. & SLEZÁRIK, A. 1983. The action of tomato and Aspergillus foetidus pectinesterases on oligomeric substrates esterified with diazomethane. Carbohydr. Res. 116: 105–111. MARKOVIČ, O., MISLOVICOVÁ, D., BIELY, P. & HEINRICHOVÁ, K. 1992. Chromogenic substrates for endo-polygalacturonase detection in gels. J. Chromatogr. 603: 243–246. MARKOVIČ, O., MOLNÁR, L. & TOMKO, J. 1954. Papierchromatographie von oxydimorphin bei Gegenwart von Morphin. Chem. Zvesti 8: 580–589. MARKOVIČ, O. & OBENDORF, R. 1998. Soybean seed pectinesterase. Seed Sci. Res. 8: 455–466. MARKOVIČ, O. & PATOČKA, J. 1977. Action of iodine on the tomato pectinesterase. Experientia 33: 711–712. MARKOVIČ, O. & REXOVÁ, Ľ. 1957. Bestimmung von Scopo¨ ¨ lamin neben einem Uberschuss von Morphin und Athylmophin. Chem. Zvesti 11: 192–197. MARKOVIČ, O. & REXOVÁ, Ľ. 1963. Untersuchung von Komponenten verschiedener Bienengiftarten. Chem. Zvesti17: 676– 684. MARKOVIČ, O. & SAJGÓ, M. 1977. Terminal amino acids, peptide map and amino acid composition of one form of tomato pectinesterase. Acta Biochim. Biophys. Acad. Sci. Hung. 12: 45–48. MARKOVIČ, O. & THEORELL, H. 1972. Rat liver alcohol dehydrogenase (RLADH), pp. 635–637. In: AKESON, A. & EHRENBERG A. (eds) Structure and Function of Oxidation Reduction Enzymes. Pergamon Press, Oxford, New York. MARKOVIČ, O., THEORELL, H. & SETHU, R. 1971. Rat liver alcohol dehydrogenase. Purification and properties. Acta Chem. Scand. 25: 195–205. MOLNÁR, L. & ZÁTHURECKÝ, L. 1994. Vývoj farmaceutického priemyslu a výskumu na Slovensku. Farm. Obzor 63: 157– 162. NELSON, D.A. & O’CONNOR, R. 1968. The venom of honeybee (Apis mellifera): free amino acids and peptides. Can. J. Biochem. 46: 1221–1226. ¨ RNVALL, STRATILOVÁ, E., DZÚROVÁ, M., MARKOVIČ, O. & JO H. 1996. An essential tyrosine residue of Aspergillus polygalacturonase. FEBS Lett. 382: 164–166. STRATILOVÁ, E., MARKOVIČ, O., DZÚROVÁ, M., MALOVÍKOVÁ, A., CAPEK, P. & OMELKOVÁ, J. 1998. The pectolytic enzymes of carrots. Biologia, Bratislava 53: 729–736. STRATILOVÁ, E., MISLOVICOVÁ, D., KAČURÁKOVÁ, M., MA¨ RNVALL, CHOVÁ, E., KOLAROVÁ, N., MARKOVIČ, O. & JO H. 1998. The glycoprotein character of multiple forms of Aspergillus niger polygalacturonase. J. Prot. Chem. 17: 173– 179. REXOVÁ, Ľ. & MARKOVIČ, O. 1963. Chemische Charakterisierung einiger niedermolekularer Komponenten des Bienengifts. Chem. Zvesti 17: 884–890. REXOVÁ-BENKOVÁ, Ľ. & MARKOVIČ, O. 1976. Pectic enzymes. Adv. Carbohydr. Chem. Biochem. 33: 323–385. ŠUBERT, J. 1992. Tajné usnesení o politické čistce v roce 1958. Nevěřili ani svým příbuzným. Lidové Noviny 31 JAN 1992, p. 10.

Unauthenticated Download Date | 9/24/15 11:35 PM