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Research paper thumbnail of Proteinase inhibitor polymorphism in the genus Vigna subgenus Ceratotropis and its biosystematic implications

Euphytica, 2002

The diversity of components for fourproteinase inhibitors found in species ofthe genus Vigna subg... more The diversity of components for fourproteinase inhibitors found in species ofthe genus Vigna subgenus Ceratotropis are described. Trypsin,chymotrypsin, subtilisin and cysteineproteinase inhibitors were analyzedby isoelectric focusing followed by thegelatin replica method. Of these proteinaseinhibitors, trypsin inhibitors showedmost polymorphism both within and betweenspecies. Many trypsin inhibitor componentswere also active to chymotrypsin. Severalaccessions had very low levels or absenceof some inhibitors, such as very low levelsof trypsin inhibitor in two accessions ofthe V. tenuicaulis and absence ofchymotrypsin inhibitors in V.grandiflora and V. subramaniana.Proteinase inhibitor polymorphism broadlyagreed with the taxonomic system for thesubgenus Ceratotropis. Based oninhibitor variation species analyzed couldbe divided into three groups whichcorresponding to sections Aconitifoliae, Angulares and Ceratotropis. Some species have verylittle variation in trypsin inhibitorsdespite wide distribution, such as, V.radiata and V. reflexo-pilosa.Accessions of other species showedconsiderable intraspecific variation fortrypsin inhibitors, such as, V.grandiflora, V. aconitifolia andV. stipulacea. Proteinase inhibitorpolymorphism provides an indication of thespecies that may have contributed a genometo the tetraploid species, V. reflexo-pilosa.

Research paper thumbnail of Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite coevolution

Euphytica, 1996

A complex of new approaches was used to study the insect-plant coevolution by using cereal pest d... more A complex of new approaches was used to study the insect-plant coevolution by using cereal pest digestive α-amylases and proteinases and their proteinaceous inhibitors in cereals. During evolution, plants can weaken the destructive affects of insect hydrolases at the expense of inhibitors in various ways, including (1) increasing the inhibitor activity and heterogeneity, (2) increasing the complexity of an inhibitor set and (3) producing highly specific insect enzyme inhibitors and bifunctional amylase/proteinase inhibitors. Insects, in turn, can decrease the influence of inhibitors by (1) increasing digestive enzyme activities, (2) by modifying a set of related activities of various digestive hydrolases, (3) by decreasing enzyme sensitivity to inhibitors and (4) by destroying inhibitors in guts by proteinases.

Research paper thumbnail of Novel proteinase inhibitors in seeds of sunflower ( Helianthus annuus L.): polymorphism, inheritance and properties

Theoretical and Applied Genetics, 2000

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinase... more A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M r from about 1,500 to 6,000.

Research paper thumbnail of The distribution of serine proteinase inhibitors in seeds of the Asteridae

Research paper thumbnail of Polymorphism and inheritance of methionine-rich 2S albumins in sunflower

Euphytica, 2003

2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell ... more 2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell 1990) from seeds of 103 sunflower (Helianthus annuus L.) accessions and analysed by SDS-PAGE, IEF andRP-HPLC. Two methionine-rich albumins SFA7 and SFA8 showed nodifferences in mobility on SDS-PAGE gels but were readily separated byRP-HPLC. Their levels also varied widely between different genotypes, inrelation to each other and as proportions of the total albumin fraction. Avariant form of SFA8 was identified which differed from the normal SFA8in its pI (6.5 compared to 6.0) and mobility on SDS-PAGE. N-terminal sequences of both the variant form of SFA8 and the majorform of SFA7 were identical to that reported previously for the normalform of SFA8 from the cultivar Hysun (Kortt et al., 1991) indicating theirstructural relatedness. Analysis of segregation in the F2 of the crossbetween lines VIR130 (variant SFA8) and VIR104 (normal SFA8) showedthat the normal and variant forms of SFA8 are encoded by alleles at asingle Mendelian locus. The levels of SFA7 and SFA8 in the seeds ofparental lines, F1 hybrids and individual F2 seeds classifiedfrom SDS-PAGE and IEF as homozygous for normal SFA8 (VIR104 type),homozygous for variant SFA8 (VIR130 type) and heterozygous (F1type) were determined by RP-HPLC. Seeds of the parental line VIR130contained 3.7% SFA7 and 19.0% SFA8 whereas seeds of VIR104contained 9.9% SFA7 and 12.8% SFA8. The F1 hybrid seedscontained a higher total amount of SFA7+8 proteins (32% comparingto 22% in each parent) which was largely accounted for by a highproportion of SFA7. The mean combined proportions of SFA7+8 in eachof the three phenotypic classes of F2 seeds were about 18–19% ofthe total. However, the combined proportions of SFA7+8 varied in therange 10–20% among the individual seeds. The ratio of SFA7 to SFA8was highest in the VIR104-type and heterozygous seeds, with the amountof SFA7 exceeding that of SFA8 in six heterozygous seeds. Theproportions of SFA7 and SFA8 were inversely correlated among individualF2 seeds. The results suggest that the amounts and proportions ofSFA7 and SFA8 are determined by genetic factors in addition toavailability of sulphur.

Research paper thumbnail of Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties

Research paper thumbnail of Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms

Research paper thumbnail of Proteinase inhibitor polymorphism in the genus Vigna subgenus Ceratotropis and its biosystematic implications

Euphytica, 2002

The diversity of components for fourproteinase inhibitors found in species ofthe genus Vigna subg... more The diversity of components for fourproteinase inhibitors found in species ofthe genus Vigna subgenus Ceratotropis are described. Trypsin,chymotrypsin, subtilisin and cysteineproteinase inhibitors were analyzedby isoelectric focusing followed by thegelatin replica method. Of these proteinaseinhibitors, trypsin inhibitors showedmost polymorphism both within and betweenspecies. Many trypsin inhibitor componentswere also active to chymotrypsin. Severalaccessions had very low levels or absenceof some inhibitors, such as very low levelsof trypsin inhibitor in two accessions ofthe V. tenuicaulis and absence ofchymotrypsin inhibitors in V.grandiflora and V. subramaniana.Proteinase inhibitor polymorphism broadlyagreed with the taxonomic system for thesubgenus Ceratotropis. Based oninhibitor variation species analyzed couldbe divided into three groups whichcorresponding to sections Aconitifoliae, Angulares and Ceratotropis. Some species have verylittle variation in trypsin inhibitorsdespite wide distribution, such as, V.radiata and V. reflexo-pilosa.Accessions of other species showedconsiderable intraspecific variation fortrypsin inhibitors, such as, V.grandiflora, V. aconitifolia andV. stipulacea. Proteinase inhibitorpolymorphism provides an indication of thespecies that may have contributed a genometo the tetraploid species, V. reflexo-pilosa.

Research paper thumbnail of Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite coevolution

Euphytica, 1996

A complex of new approaches was used to study the insect-plant coevolution by using cereal pest d... more A complex of new approaches was used to study the insect-plant coevolution by using cereal pest digestive α-amylases and proteinases and their proteinaceous inhibitors in cereals. During evolution, plants can weaken the destructive affects of insect hydrolases at the expense of inhibitors in various ways, including (1) increasing the inhibitor activity and heterogeneity, (2) increasing the complexity of an inhibitor set and (3) producing highly specific insect enzyme inhibitors and bifunctional amylase/proteinase inhibitors. Insects, in turn, can decrease the influence of inhibitors by (1) increasing digestive enzyme activities, (2) by modifying a set of related activities of various digestive hydrolases, (3) by decreasing enzyme sensitivity to inhibitors and (4) by destroying inhibitors in guts by proteinases.

Research paper thumbnail of Novel proteinase inhibitors in seeds of sunflower ( Helianthus annuus L.): polymorphism, inheritance and properties

Theoretical and Applied Genetics, 2000

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinase... more A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M r from about 1,500 to 6,000.

Research paper thumbnail of The distribution of serine proteinase inhibitors in seeds of the Asteridae

Research paper thumbnail of Polymorphism and inheritance of methionine-rich 2S albumins in sunflower

Euphytica, 2003

2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell ... more 2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell 1990) from seeds of 103 sunflower (Helianthus annuus L.) accessions and analysed by SDS-PAGE, IEF andRP-HPLC. Two methionine-rich albumins SFA7 and SFA8 showed nodifferences in mobility on SDS-PAGE gels but were readily separated byRP-HPLC. Their levels also varied widely between different genotypes, inrelation to each other and as proportions of the total albumin fraction. Avariant form of SFA8 was identified which differed from the normal SFA8in its pI (6.5 compared to 6.0) and mobility on SDS-PAGE. N-terminal sequences of both the variant form of SFA8 and the majorform of SFA7 were identical to that reported previously for the normalform of SFA8 from the cultivar Hysun (Kortt et al., 1991) indicating theirstructural relatedness. Analysis of segregation in the F2 of the crossbetween lines VIR130 (variant SFA8) and VIR104 (normal SFA8) showedthat the normal and variant forms of SFA8 are encoded by alleles at asingle Mendelian locus. The levels of SFA7 and SFA8 in the seeds ofparental lines, F1 hybrids and individual F2 seeds classifiedfrom SDS-PAGE and IEF as homozygous for normal SFA8 (VIR104 type),homozygous for variant SFA8 (VIR130 type) and heterozygous (F1type) were determined by RP-HPLC. Seeds of the parental line VIR130contained 3.7% SFA7 and 19.0% SFA8 whereas seeds of VIR104contained 9.9% SFA7 and 12.8% SFA8. The F1 hybrid seedscontained a higher total amount of SFA7+8 proteins (32% comparingto 22% in each parent) which was largely accounted for by a highproportion of SFA7. The mean combined proportions of SFA7+8 in eachof the three phenotypic classes of F2 seeds were about 18–19% ofthe total. However, the combined proportions of SFA7+8 varied in therange 10–20% among the individual seeds. The ratio of SFA7 to SFA8was highest in the VIR104-type and heterozygous seeds, with the amountof SFA7 exceeding that of SFA8 in six heterozygous seeds. Theproportions of SFA7 and SFA8 were inversely correlated among individualF2 seeds. The results suggest that the amounts and proportions ofSFA7 and SFA8 are determined by genetic factors in addition toavailability of sulphur.

Research paper thumbnail of Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties

Research paper thumbnail of Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms