Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans

A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests.     

Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.     

Purification and properties of a lipid acyl-hydrolase from potato tubers.

1. A pure lipid acyl-hydrolase was prepared from potato tubers by acetone precipitation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography, and by electrofocusing. 2. The purified enzyme was an acidic protein of pI 5.0 and molecular weight of about 70 000. Km values were 0.38 mM for monogalactosyldiacylglycerol and 1.7 mM for phosphatidylcholine. 3. The hydrolytic activity of the enzyme on different substrates was determined. The relative rates were acylsterylglucoside greater than monogalactosyldiacylglycerol greater than monogalactosylmonoacylglycerol greater than digalactosyldiacylglycerol greater than diagalactosylmonoacylglycerol, while the rates for phospholipids were lysophosphatidylcholine greater than phosphatidylcholine greater than lysophosphatidylethanolamine greater than phosphatidylethanolamine. 4. Analyses of enzymatic hydrolysis products suggested that a single enzyme had both galactolipase and phospholipase activities, and for the phospholipids it showed activities similar to phospholipase B and glycerylphosphorylcholine diesterase. 5. A competitive relation was found between monogalactosyldiacylglycerol and phosphatidylcholine as substrates of the enzyme, indicating that the active sites for both substrates may be the same. 6. It was suggested that histidine and probably serine residues were important to the enzymic activity, and that a tyrosine residue might be involved in the activity as an accessory component.     

Purification and multiple forms of phosphoglucomutase from potato tubers

Two fractions (Peaks I and II) having phosphoglucomutase activitywere separated by DEAE-cellulose chromatography of the crudeenzyme preparation from potato tubers. Upon separate rechromatographyunder the same conditions, they were respectively eluted atthe same positions as initially eluted. Incubation of PeaksI and II with glucose 1,6-bisphosphate which should cause conversionof the dephosphorylated form to the phosphorylated form causedno change in their eluting positions in rechromatography. Theratio of their contents in the crude extracts were fairly constantamong different samples of potato tubers. Peak I was furtherseparated into three fractions on isoelectric focusing. Theresulting main fraction (Peak I_a) was purified 1,800-fold overthe crude extract with a 9% yield. It was homogeneous as judgedby polyacrylamide gel electrophoreses in the absence and presenceof sodium dodecyl sulfate. Crude extracts from peas and broadbeans each contain a single species of phosphoglucomutase whichcorresponds on the chromatograph to the Peak II enzyme frompotato tubers. It is suggested that phosphoglucomutase frompotato tubers contains at least two, possibly four, differentprotein species. (Received December 22, 1977; )     

Purification and properties of ascorbate free-radical reductase from potato tubers

Ascrobate free-radical reductase (EC 1.6.5.4) from potato tubers was purified to apparent homogencity by a method which included ammonium-sulfate precipitation, gel filtration and chromatography on diethylaminoethyl cellulose and hydroxylapatite. Gel filtration and gel electrophoresis showed that the purified enzyme was monomeric with a molecular weight of about 42 000. Enzyme activity was heat lable and severely inhibited by thiol reagents. The K_m values for enzyme substrates were estimated.Abbreviations AFR ascorbate free radical - AsA ascorbic acid - DE-32(52) diethylaminoethyl cellulose - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine     

Developing a biocontrol strategy to protect stored potato tubers from infestation with potato tuber moth species in the Andean region

Heavy infestations of stored potato (Solanum tuberosum L.) tubers by the two potato tuber moth species Symmetrischema tangolias (Gyen) and Tecia solanivora (Povolny) frequently occur in Andean potato‐growing regions of Ecuador. The aim of the study was to develop a biological control strategy for both species using powder formulations made of inert substances, Phthorimaea operculella (Zeller) granulovirus (PhopGV) and Bacillus thuringiensis Berliner subsp. kurstaki (Btk). The LC₅₀ of PhopGV on T. solanivora was 0.33 LE/L, and Btk caused 82.7% mortality at a concentration of 100 g/L in bioassays. The efficacy of talcum, kaolin, calcium carbonate and sand ranged between 76.2% and 98.7%. Calcium carbonate was highly effective to control both species; however, its efficacy was affected by the relative humidity and dropped to 55.4% at relative humidity of 100%. PhopGV at concentrations of five larvae equivalents (LE) per kg kaolin and Btk at a concentration of 60 g Btk/kg talcum caused 95.7% and 88.1% mortality of T. solanivora, respectively. In storage experiments, the efficacy of calcium carbonate alone and in combination with PhopGV (20 LE/kg) and Btk (15 g/kg) caused 95.0–99.8% mortality of T. solanivora in all treatments and reduced infestation on potato tubers by 83.6%–91.0%. In the case of S. tangolias, Btk significantly increased mortality to 96.5% compared with calcium carbonate alone and reduced tuber infestation by 83.4%. Storage of potato tubers in thin layers enhanced the efficacy of the calcium carbonate treatment compared with storage in bags. It was concluded that calcium carbonate alone seems to be appropriate for the control of T. solanivora, and an addition of 15 g Btk/kg would improve the control of S. tangolias. It is suggested to test these new formulations under on‐farm storage conditions.     

Primary structure of a 21-kD protein from potato tubers

A 21-kD protein isolated earlier from potato tubers (Solanum tuberosum L.) has two isoforms, with pI 6.3 and 5.2, which were separated by fast protein ion-exchange chromatography on a Mono Q column. The primary structures of the two forms consisted of 187 and 186 amino acid residues. Both isoforms are composed of two polypeptide chains, designated A and B, linked by a single disulfide bond between Cys-146 of the A chain and Cys-7 of the B chain. The amino acid sequences of the A chains of the two forms, consisting of 150 residues each, differ in a single amino acid residue at position 52 (Val --> Ile), while the B chains, containing 37 and 36 residues, respectively, have substitutions at nine positions (Leu-8 --> Ser-8, Lys-25--Asp-26 --> Asn-25--Glu-26, Ile-31--Ser-32 --> Val-31--Leu-32, Lys-34--Gln-35--Val-36--Gln-37 --> Gln-34--Glu-35--Val-36). Both isoforms form stable inhibiting complexes with human leukocyte elastase and are less effective against chymotrypsin and trypsin.     

Purification and properties of an aspartic protease from potato tuber that is inhibited by a basic chitinase

A protease was isolated from potato ( Solanum tuberosum L. cv. Huinkul) tuber disks after 24 h of aeration when proteolysis is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration, it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it had a pH optimum between 4 and 5 and it was inhibited by pepstatin. Partial homology with other plant aspartic proteinases was observed in two sequenced tryptic fragments. It binds to Sepharose-concanavalin A and can be eluted with α -methyl mannoside, indicating that it is possibly glycosylated. Unlike other aspartic proteinases from Solanaceae that degrade pathogenesis-related proteins, it is unable to cleave a basic chitinase from potato. Moreover, this aspartic protease is strongly inhibited by the basic chitinase; the 50% inhibition is obtained when the molar ratio approaches 1, the same as with pepstatin. The interaction between this aspartic protease and a new type of endogenous inhibitor may be an interesting starting point to study the regulation of these aspartic proteases during stress.     

Purification and properties of l(+)-lactate dehydrogenase from potato tubers

1. A purification of l(+)-lactate dehydrogenase is described. 2. The final preparation is active with NADH and NADPH and with a number of keto acids, but evidence is presented to support the view that a single enzyme is involved. 3. NAD(+) showed product inhibition, but at slightly acid pH values there was evidence of co-operative binding. 4. At acid pH values ATP was a potent inhibitor and appears to be an allosteric effector. At neutral or alkaline pH values ATP behaved as a weak competitive inhibitor. 5. The physiological significance of inhibition by ATP is discussed.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.     

Purification and characterization of an a type phospholipase from potato and its effect on potato mitochondria

A potato (Solanum tuberosum) phospholipid acyl-hydrolase, which - in the pH range 7.5 to 8.5—is at least 10,000 times more effective with phospholipids than with galactolipids, has been purified and characterized. It is a soluble enzyme readily distinguished from a neutral lipid lipase and a third lipid acyl-hydrolase which, while acting on phospholipid, shows a decided preference for glyceryl monoolein. The phospholipase in question has a pH optimum of 8.5, is stimulated by Ca²⁺ at pH above 7.5 and inhibited by Ca²⁺ at lower pH, is not dependent on detergents although stimulated by Triton X-100 to a moderate extent, and remains very active at temperatures close to zero. The phospholipids of intact potato mitochondria are highly susceptible to degradation by potato phospholipase, and it is suggested that this enzyme is involved in the extensive lipid breakdown which occurs in fresh potato slices following cutting, and in the deterioration of mitochondria during their preparation and aging.     

Increase in ribonuclease activity following mechanical damage to leaf and tuber tissues of Solanum tuberosum L.

Summary Major increases occurred in the capacity of damaged potato leaf and tuber tissues to hydrolyse ribonucleic acid whilst relatively minor increases were found in the activity of acid phosphomonoesterase and acid phosphodiesterase. Partial purification of homogenates by gel filtration on Sephadex G-100 revealed that much of the increased capacity to degrade ribonucleic acid following damage was due to increased ribonuclease activity. Although appreciable differences in the elution patterns of tuber homogenates subjected to gel filtration were observed before and after the breaking of dormancy the increased ribonuclease activity following damage was a constant feature. Actinomycin D had a relatively small effect on preventing these increases in phosphate-ester hydrolase activities whilst the effect of cycloheximide was very pronounced. Isopycnic equilibrium centrifugation experiments, using deuterium oxide as a density label, provided no evidence that the increased enzyme activity following damage was due to synthesis of new enzyme.     

Purification and characterization of the 22-kilodalton potato tuber proteins

Three abundant proteins of approximate molecular masses of 22, 23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass forms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified proteins, and the aminoterminal amino acid sequences were determined for these three proteins. All three proteins have identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA.     

Identification of a yeast ribonuclease H as an Sm antigen

We have isolated a 55-kDa enzyme from Saccharomyces cerevisiae on the basis of its ability to hydrolyze specifically the RNA moiety of RNA/DNA hybrids [RNase H(55)]. Remarkably, monospecific anti-[RNase H(55)] antibodies revealed that the protein associates with several small RNAs, including some of the essential yeast spliceosomal snRNAs. Moreover, immunoprecipitation as well as immunoblotting experiments demonstrated that the yeast enzyme reacts (a) with human anti-Sm autoantisera, (b) with a monoclonal antibody specific for the human snRNP proteins B/B', but (c) not with U1-ribonucleoprotein-specific autoantibodies. These results disclosed a hitherto unexpected degree of evolutionary conservation in snRNP protein structure between yeast and man. Additionally, our findings suggested a re-evaluation of the enzymatic mechanism of RNases H which recognize both RNA and RNA/DNA hybrids.     

An immunochemical study of antigen expression in potato spindle tuber viroid (PSTV) - infected tomato leaves and calluses

A polyspecific antiserum against protein extracted from PSTV-infected tomato leaves was prepared and the IgGs were separated by affinity chromatography on a beaded cellulose adsorbent with an immobilized “healthy” antigen. The antibody not adsorbed entered into a preferential reaction with the antigen from PSTV-infected leaves as estimated by an enzyme-linked immunosorbent assay. The immunochemical reactions did not significantly exceed the control background, if antigens from tomato leaves infected with potato viruses X, Y and M were analyzed. By immunoblot technique we revealed, however, that several antigens not detected in healthy leaves appeared in the leaves infected either with PSTV or with viruses X and M. An accumulation of a major antigen having a molecular mass of about 70 kDa was observed in viroid-infected leaves only, suggesting the specificity for viroid infection. The antigen was found not to be an alkaline endoproteinase - the pathogenesis-related protein P-69. Some antigens with molecular masses approximately 38.0, 23.7 and 22 kDa, which occurred in PSTV-infected leaves and in healthy calluses, were not detectable in PSTV-infected calluses. No reaction exceeding the control level was observed using enzyme-linked immunosorbent assay for antigens from silver nitrate-treated tomato leaves, although such leaves showed symptoms similar to that caused by viroids.     

Purification and characterization of 3-hydroxy-3-methylglutaryl CoA reductase from potato tubers

3-Hydroxy-3-methylglutaryl coenzyme A reductase (NADPH) was solubilized by trypsin digestion from sliced potato tuber microsomes, and purified to apparent homogeneity in the absence of detergent with a recovery of 1.8%. The enzyme had a specific activity of 7,910 nmol of mevalonate formed per min per mg of protein. On molecular-sieving high-performance liquid chromatography, the activity was coincident with the single protein peak corresponding to a molecular weight of approximately 110 kDa. On SDS-polyacrylamide gel electrophoresis, the purified enzyme showed only one protein staining band corresponding to a molecular weight of approximately 55 kDa. The apparent Km value for S-HMG-CoA was 6.4 microM and that for NADPH was 25 microM.     

The significance of tuber damage and inoculum concentration of Phoma exigua var. foveata in the development of gangrene in stored potato tubers

The susceptibility to gangrene infection of wounds of various shapes and depths on potato tubers was studied by inflicting wounds using differently-shaped brass teeth and rods of different diameters. Inoculating wounds with spore suspensions or damaging tubers which had been previously contaminated with Phoma exigua var. foveata or which had been recently lifted from plots of field experiments showed that wounds in which tissue was crushed were most susceptible to infection. Over a wide range of inoculum concentrations and in experiments using several different cultivars the incidence of infection of any wound type was compared to that of the standard severe cut and crush wound. Using a probit transformation a linear relationship was established, the slope of the line indicating the relative susceptibility of the wound. In 1977 and 1978, crops of cv. Pentland Dell were surveyed for damage incidence, inoculum and inoculum potential on arrival at a commercial bulk store. Nets of tubers buried among the tuber bulk were recovered after storage and gangrene incidence compared with damage and inoculum assessments. Inoculum potential and incidence of severe damage both influenced disease development but damage incidence was of greater importance, showing that priority should be given to decreasing damage and to curing to promote rapid wound healing in endeavours to control the disease.     

Changes in potato tuber invertase and its endogenous inhibitor after slicing, including a study of assay methods

The increase in the invertase activity of extracts from freshly cut potato (Solanum tuberosum L.) by “foaming,” caused by selective denaturation of an endogenous invertase inhibitor, did not occur in extracts made from thin disks 2 days after slicing. Rather, foaming such extracts decreased invertase activity. Apparently, the inhibitor disappeared after slicing, and the enzyme became more labile to foaming. Such disappearance of inhibitor could account for up to 15% of the dramatic increase in total invertase activity that had occurred within 2 days after slicing. The difference between extracts from 0-day and 2-day slices was mainly in the first of two peaks of invertase activity eluted from diethylaminoethyl-cellulose columns. This peak was increased by foaming 0-day extracts, but even when foamed was much smaller than in 2-day extracts. The apparent loss in inhibitor was not caused by a decreasing susceptibility of the enzyme to the inhibitor. Both the increase in total invertase activity and the apparent loss of inhibitor after slicing were partially blocked by actinomycin D and completely blocked by cycloheximide.