A releationship between protein-degradation rates <i>in vivo</i>, isoelectric points, and molecular weights obtained by using density labelling

Acton, G. John; Gupta, S. C.

doi:10.1042/bj1840367

Cited by 19 publications

(6 citation statements)

References 63 publications

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“…The degradation of both subunits of RuBPCase followed different kinetics, the larger subunit being more rapidly degraded than the small subunit, which needed incubation times longer than 4 h to undergo detectable degradation (data not shown). This could be explained by the different mol wt of both subunits, in accord with the general scheme proposed for other proteolytic systems (1,10 have failed to detect such intermediates. As pointed out by Muller et al (23), the failure to demonstrate intermediates in the degradation processes may be explained by the fact that they might be short-lived or unrecognizable by antibodies.…”

Section: Resultsmentioning

confidence: 52%

Pathogenesis-Related Proteins of Tomato

Vera

Conejero²

1988

Plant Physiol.

127

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An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv "Rutgers") leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pl around 9.0.The term PR2 proteins refers to a group of plant encoded proteins whose synthesis is induced when plants react against infections by viroids, viruses, and several other pathogens as well as stress situations such as those produced by chemical treatments, plasmolysis, and even natural senescence of the plant (29).Infection of tomato plants by CEV results in a remarkable increase of 10 PR proteins whose synthesis seems to be mediated by ethylene (13).Although considerable information has been obtained regarding the appearance, properties, amino acid, and nucleotide sequences, and regulation of their synthesis, no specific function or biological activity has been assigned to PR proteins aside from their involvement in protection phenomena (4,7,8,15,16,19,20,29).In this paper, we show that one of the major PR proteins induced by CEV in tomato plants, previously reported as P-69 (13), is an endoproteinase. We also speculate about the possible role of P-69 in the infected tissue. MATERIALS AND METHODS

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Section: Resultsmentioning

confidence: 52%

Pathogenesis-Related Proteins of Tomato

Vera

Conejero²

1988

Plant Physiol.

127

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“…Because of the difference in molecular weight of the two subunits, it oiight be expected that their degradation (i.e. the disappearance of the SDS-PAGE bands) should not occur at the same rate, the larger subunit being more rapidly hydrolyzed than the smaller (Dice andGoldberg 1975, Acton andGupta 1979). Our experiments showed just the opposite, at least at pH 3.7 and 4.5, indicating a preference of the proteolytic enzymes for the small subunits.…”

Section: Diseussionmentioning

confidence: 58%

Proteolysis of ribulose‐1,5‐bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Garcı́a-Martı́nez¹,

Moreno²

1986

Physiologia Plantarum

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Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) has been purified from orange [Citrus sinensis (L.) Osbeck cv. Washington Navel] leaves using sucrose gradient centrifugation in a fixed angle rotor. Following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), two major bands corresponding to the two subunits of RuBP carboxylase were found. The large subunit coincided with the polypeptide band that has been previously reported to be preferentially mobilized during the spring and summer flush periods. The degradation of RuBP carboxylase during autodigestion of Citrus leaf extracts, investigated by SDS‐PAGE, occurred mainly at acidic (2.5‐5.5) pH. The two subunits showed differences in the rate of degradation, the smaller being more rapidly hydrolyzed than the larger. At least four proteolytic activities were identified by means of inhibitor experiments: 1) a pepstatin A‐sensitive activity that acts on both RuBP carboxylase subunits, 2) a mercurial (p‐hydroxymercuribenzoate and p‐chloromercuriphenylsulfonate)‐sensitive activity that degrades only the small subunit, 3) an EDTA‐sensitive activity that hydrolyzes both the large and small subunits, and 4) a mercurial‐stimulated activity that acts only on the large subunit. It is suggested that the last two proteases may be responsible for the degradation of RuBP carboxylase observed in vivo during the periods of mobilization of leaf protein in Citrus.

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“…Evidence supporting the existence of a correlation between the physical properties of proteins (charge and size) and their rates of degradation has been reviewed by Goldberg and St. John (17). A number of experiments, designed to test these correlations in plants, produced results broadly consistent with the general hypothesis (1,10). However, more recent experiments, employing different methods, have suggested that, if the correlations exist, they are at best weak (8,9).…”

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confidence: 83%

“…Two batches of Lemna fronds were grown in complete medium (750 ml) containing either 1 The results of this experiment are shown in Figure 7. The pattern of protein degradation in darkness is significantly different from that observed in the light.…”

Section: Uferreira and Daviesmentioning

confidence: 99%

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase

Ferreira¹,

Davies²

1987

Plant Physiol.

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Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modifilcation of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.A great deal of evidence has been marshaled to support the view that the key enzyme of photosynthesis, RuBPCase' (EC 4.1.1.39), undergoes little or no degradation prior to leaf senescence. The strongest evidence that RuBPCase does not degrade while it is being synthesized has been presented by Huffaker and his colleagues (19,29). The same group has shown that, during the dark-induced senescence of detached leaves of barley, the initial loss of protein is almost entirely due to the degradation of RuBPCase (28). This behavior has also been observed in the dark-induced senescence of attached wheat leaves (37, 38). The stability of RuBPCase prior to senescence, and its rapid degradation during senescence, has led to its classification as a leaf storage protein ( 19,21 is the radioactivity present in protein immediately after labeling with a radioactive amino acid and P(,) is the radioactivity remaining after time t. This ratio can readily be transformed to give the rate constant of degradation (KD) by means of the equation for first order decay KDt = ln(P(o)/P(,)).Alternatively, degradation can be expressed as a half-life t(o.5) t(O.5) = ln 2/KD. For reasons presented in the "Discussion" we prefer to use the direct isotope ratio, rather than the logarithmic transform.The double-label method (2) has been widely used to test the proposition that t...

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A releationship between protein-degradation rates in vivo, isoelectric points, and molecular weights obtained by using density labelling

Cited by 19 publications

References 63 publications

Pathogenesis-Related Proteins of Tomato

Pathogenesis-Related Proteins of Tomato

Proteolysis of ribulose‐1,5‐bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase

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