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Biomedical subjects

C A Ryan

Publications and source records attributed to C A Ryan.

At least 235 records · Page 13Linked to original sources
PubMed1981

Proteinase inhibitor-inducing factor activity in tomato leaves resides in oligosaccharides enzymically released from cell walls.

The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-alpha-1,4-polygalacturonase digestion of a pectic polysaccharide (M(r) 5000-10,000) isolated from tomato leaves. Active oligosaccharides were also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either endogenous or exogenous endopolygalacturonases at a wound or infection site, may have hormone-like roles in regulating plant defense responses in unwounded tissues many centimeters away from the site of release.

Journal Article↗
PubMed1981

Regulation of Proteinase Inhibitor Synthesis in Tomato Leaves : IN VITRO SYNTHESIS OF INHIBITORS I AND II WITH mRNA FROM EXCISED LEAVES INDUCED WITH PIIF (PROTEINASE INHIBITOR INDUCING FACTOR).

Messenger RNA was isolated from young excised tomato leaves, induced to accumulate proteinase Inhibitors I and II with the proteinase inhibitor inducing factor (PIIF), and translated in vitro in a rabbit reticulocyte lysate system. Translatable messenger RNAs specific for Inhibitors I and II were present in PIIF-induced leaves but were not present without PIIF induction. The nascent in vitro-synthesized inhibitors migrated with an apparent molecular weight 2,000 to 3,000 daltons larger than that of the two inhibitors isolated from leaves. The molecular weights of the preinhibitors were identical whether translated from mRNA from PIIF-induced leaves or translated from mRNA isolated from wounded leaves. Incubation of excised PIIF-induced plants in CO(2)-free air doubled the rate of in vivo synthesis of Inhibitor I over that in normal air (Ryan CA 1977 Biochem Biophys Res Commun 77: 1004-1008) but did not affect the rate of in vivo Inhibitor II accumulation. The rate of incorporation of (35)SO(4) (2-) into soluble proteins was 70% less when leaves were incubated in CO(2)-free air rather than normal air. Messenger RNA isolated from PIIF-induced plants incubated in the presence or absence of CO(2) was translated in vitro. The amount of in vitro-translatable mRNA present for each inhibitor (per microgram total mRNA) was the same in leaves incubated in either atmosphere. Therefore, the increased rate of synthesis and accumulation of Inhibitor I in a CO(2)-free atmosphere does not appear to result from an increased level of mRNA but appears to be controlled at a posttranscriptional level.

Journal Article↗
PubMed1981

A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities.

A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-alpha-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.

Journal Article↗
PubMed1980

Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities.

The single disulfide bond in potato Inhibitor I protomers (Mr = 8,000) was reduced and alkylated in the absence of denaturants to the carboxamidomethyl cysteine derivative. Two half-cystines per mol of inhibitor protomer were modified as determined by (a) loss of two sulfhydryl groups per reduced protomer, (b) incorporation of 2 mol of [14C]iodoacetamide per protomer, and (c) amino acid analyses. The alkylated Inhibitor I retained its oligomeric structure (Mr = 40,000) and fully retained its immunological cross reactivity with anti-Inhibitor I serum. Furthermore, the inhibitory properties of modified and unmodified Inhibitor I toward chymotrypsin were identical. The stoichiometric complex between modified Inhibitor I and chymotrypsin was stable at pH 8.0 for over 72 h. The modification of the disulfide bond introduced a lability to both heat and proteolytic enzymes. Ultraviolet difference spectra and near ultraviolet circular dichroism spectra at pH 8.0, between modified and unmodified Inhibitor I, revealed only minor absorption changes due to modification. However, in circular dichroism spectra at pH 2.0, the modified inhibitor exhibited a significant loss of absorption in the region between 270 and 280 nm that was present in the unmodified inhibitor. The cumulative results of this study indicate that the single disulfide bond in Inhibitor I, which forms a rather large disulfide loop between residues 5 and 51, is not important at neutral pH for maintaining major structural conformations which affect immunological or inhibitory activities, but the disulfide bond does impose restraints on the protein which stabilize it toward thermal denaturation and proteolysis. The susceptibility of the reduced inhibitor to plant sulfhydryl enzymes may have some importance in in vivo degradation.

Alkylation↗
PubMed1980

In vitro synthesis of pre-proteins of vacuolar compartmented proteinase inhibitors that accumulate in leaves of wounded tomato plants.

Two proteinase inhibitor proteins that are compartmented in leaf vacuoles (lysosomes) were synthesized in vitro. mRNA was isolated from 17-day-old expanding tomato leaves by extraction with chaotropic buffers followed by chromatography on oligo(dT)-cellulose and was translated with a rabbit reticulocyte lysate system. Preparations of mRNA from leaves of both wounded plants and unwounded plants directed the incorporation of equivalent amounts of label into trichloroacetic acid-precipitable proteins. Only mRNA from leaves of wounded plants directed label into proteins that could be immunoprecipitated with rabbit IgG specific for either inhibitor I or inhibitor II. These results indicate that the wound-induced accumulation of proteinase inhibitors I and II in leaf vacuoles is a result of the presence of translatable mRNA species not present in leaves of unwounded plants. Gel electrophoresis of the immunoprecipitates in NaDodSO(4)/urea/polyacrylamide gels revealed that inhibitors I and II were translated in vitro as precursors about 2000 daltons larger than the inhibitors found in leaves. The presence of the additional polypeptide sequences in the newly synthesized inhibitors indicates that the inhibitors are processed either during or after synthesis, and the presequences may be signal peptides that are part of the process of inhibitor transport into the vacuolar compartments of tomato leaf cells.

Journal Article↗
PubMed1977

Disulfide crosslinks and the specificity of protein turnover in plants.

Studies of the protein metabolism of detached tomato leaves, hormonally induced to accumulate proteinase inhibitors, have indicated that the state of oxidation of protein-bound half-cystine residues may be a principal parameter affecting in vivo and in vitro stability of leaf proteins. Induced leaves exhibited a general specificity of intracellular protein degradation directed towards the preferential hydrolysis of proteins having free-sulfhydryl residues. Proteins having disulfide cross-linkages, including the proteinase inhibitors, were markedly stable to in vivo degradation, and as a result, accumulated. These results provide a precedence for a cellular protein selection process, resulting from a directed specificity of intracellular protein degradation, which is focused on a particular protein structural parameter.

Cysteine↗
PubMed1977

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera.

Proteinase inhibitor-inducing factor (PIIF)-induced accumulation of trypsin inhibitory activity was assayed in leaves of 23 species of plants representing 10 agriculturally important genera. Inhibitory activity was assayed in extracts from attached leaves or from excised leaves supplied through the cut petioles for 30 minutes with extracts containing the wound hormone PIIF, obtained from either tomato leaves or from the leaves of each plant under study. During subsequent incubation in light for 72 hours, PIIF-induced trypsin inhibitory activity accumulated in significant quantities in 10 of the 23 species. Alfalfa accumulated the highest levels of inhibitory activity (340 mug trypsin inhibited/ml leaf juice), followed by tobacco, tomato, potato, strawberry, cucumber, squash, clover, broadbean, and grape. It is suggested that the inhibitors might be classed as allelochemics that are present in certain plants and not others in response to environmental pressures during their evolution.

Journal Article↗
PubMed1977

Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles.

Proteinase inhibitor I has been identified and quantified in isolated vacuoles from tomato (Lycopersicon esculentum) leaves induced to accumulate inhibitors either by wounding or by supplying excised leaves with the wound hormone, proteinase inhibitor-inducing factor. Proteinase inhibitor II was also identified in the vacuoles but not quantified. Control vacuoles were prepared from unwounded plants that did not contain inhibitors. Vacuole to leaf cell ratios of inhibitors, chlorophyll, and several vacuolar and cytoplasmic enzymes were determined. The inhibitors were found almost entirely in the vacuoles. Acid phosphatase was located in control leaf vacuoles, but was found in both vacuoles and cytoplasm in induced leaves. Carboxypeptidase, induced by wounding, was found distributed between the vacuoles and cytoplasm of induced leaves. Low vacuole to leaf cell ratios of three cytoplasmic markers, triosephosphate isomerase, catalase, and chlorophyll, indicated that the isolated vacuoles were relatively free of intact protoplasts and cell debris.

Journal Article↗
PubMed1976

Specificity of protein turnover in tomato leaves. Accumulation of proteinase inhibitors, induced with the wound hormone, PIIF.

Detached tomato leaves, supplied with the proteinase inhibitor inducing factor (PIIF) and incubated with water under constant light, exhibited a specificity of intracellular protein turnover directed toward the selective accumulation of heat-stable proteins having disulfide corss-linkages. Approximately 70% of the accumulated proteins could be accounted for in two proteinase inhibitors rich in disulfide links. The accumulation of proteins containing disulfides was accompanied by a net loss in total leaf protein, mainly of heat-precipitable proteins having free sulfhydryl residues. Relative rates of synthesis of --S--S-- proteins and --SH proteins were assessed by comparing rates of incorporation of isotope into the inhibitor proteins and noninhibitor leaf proteins. Although the inhibitors represented about 12% of total leaf protein after 71 h of induction, only about 2% of total protein synthesis was directed toward inhibitor synthesis during incubation of induced leaves. The marked stability of inhibitors, and other disulfide proteins against degradation in vivo, appeared to be a major factor providing for their selective accumulation. It was concluded that the state of oxidation of protein-bound half-cystine residues may be a principle parameter influencing the susceptibility of leaf proteins to degradation in vivo.

Binding Sites↗
PubMed1976

Proteinase inhibitor II from potatoes: isolation and characterization of its protomer components.

Proteinase inhibitor II, an inhibitor of chymotrypsin and trypsin, is a heat-stable protein with a dimeric molecular weight of 21 000 that is a component of Russet Burbank potato tubers. Four monomeric isoinhibitor species of molecular weight 10 500 comprise inhibitor II and were isolated by chromatography on phosphocellulose in 8 M urea. Upon removal of the urea, each monomeric species dimerized to yield homogeneous dimers. The three major protomer species, called B, C, and D, and their homogeneous dimers were further characterized. They have similar molecular weights and amino acid compositions, and each has an N-terminal alanine residue. Dimers of purified protomers B, C, and D exhibited full cross-reactivities with each other in immunological double-diffusion assays. Reconstituted dimers possess two binding sites for bovine alpha-chymotrypsin, indicating that each monomer possesses one binding site for this enzyme. Significant differences were noted among the reconstituted dimers in their isoelectric points, immunoelectrophoretic mobilities, ion-exchange properties, and their inhibitory reactivities against trypsin. The properties of the inhibitor II dimeric species are similar but not identical to inhibitors IIa and IIb reported from Japanese potatoes (variety "Danshaku-Imo"), indicating the existence of intervarietal, as well as intravarietal, differences among potato tuber inhibitor II isoinhibitors.

Amino Acids↗
PubMed1976

Homologous inhibitors from potato tubers of serine endopeptidases and metallocarboxypeptidases.

A potent polypeptide inhibitor of chymotrypsin has been purified from Russett Burbank potatoes. The inhibitor has no effect on bovine carboxypeptidases A or B but exhibits homology with a carboxypeptidase inhibitor that is also present in potato tubers. The chymotrypsin inhibitor has a molecular weight of approximately 5400 as estimated by gel filtration, amino acid analysis, and titration with chymotrypsin. The polypeptide chain consists of 49 amino acid residues, of which six are half-cystine, forming three disulfide bonds. Its size is similar to that of the carboxypeptidase inhibitor, which contains 39 amino acid residues and also has three disulfide bridges. In immunological double diffusion assays, the chymotrypsin inhibitor and the carboxypeptidase inhibitor do not crossreact; however, automatic Edman degradation of reduced and alkylated derivatives of the chymotrypsin inhibitor, yielding a partial sequence of 18 amino acid residues at the NH2-terminus, reveals a similarity in sequence to that of the carboxypeptidase inhibitor. Thus, inhibitors directed toward two distinct classes of proteases, the serine endopeptidases and the metallocarboxypeptidases, appear to have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1976

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato.

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 mug of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.Immunological and electrophoretic properties of inhibitor I from tobacco crown gall tumor, callus, etiolated, and variegated tissues were compared. Agar immunodiffusion assays showed no apparent differences among precipitin reaction lines between inhibitor I of tumor, callus, variegated, and etiolated tissues. The immunoelectrophoretic mobilities of inhibitor I of tumor, variegated, and etiolated tissues were the same, but differed from that of either normal or crown gall callus tissues. These results suggest that different isoinhibitors of inhibitor I could account for the observed differences in electrophoretic mobilities, or that modification of the inhibitor has occurred sometime during, or after, its synthesis.

Journal Article↗