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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

GroEL140, a mutant Escherichia coli chaperonin unable to support bacteriophage lambda head assembly, was purified to near homogeneity and compared to wild type GroEL (cpn60). GroEL140 exhibited a 1.5-fold lower ATPase activity relative to the wild type protein. The hydrolysis of ATP by both polypeptides was fully inhibited by an excess of ATP gamma S and partially inhibited by ADP and 5'-adenylylimidodiphosphate, suggesting that adenine nucleotides display different affinities for the ATP binding site of chaperonins. GroEL140 was more sensitive to trypsin digestion compared to wild type GroEL indicating that the mutation destabilized the conformation of the mutant. The proteolytic susceptibility of both chaperonins was similarly enhanced upon addition of ATP, ADP or non-hydrolyzable ATP analogs, providing evidence (i) of a conformational change in the chaperonin structure which is likely to drive the protein discharge process, and (ii) that hydrolysis of ATP is not required to achieve topological modifications. GroEL140 retained its ability to bind non-native ribulose bisphosphate carboxylase/oxygenase (Rbu-P2-carboxylase), but released bound proteins upon addition of ATP and GroES (cpn 10) 6-7-fold less efficiently compared to GroEL. This functional defect was shown to be related to a suboptimal, but not an absence of, interaction with GroES since (i) GroEL140 and GroES were unable to form a complex isolatable by size exclusion chromatography, and (ii) increasing the incubation time or the concentration of GroES enhanced the amount of refolded Rbu-P2-carboxylase discharged from GroEL140-Rbu-P2-carboxylase binary complexes. Pulse-chase experiments involving a double immunoabsorption technique confirmed that Rbu-P2-carboxylase remained associated two times longer with GroEL140 than with GroEL in vivo.
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PMID:A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides. 135 Jul 86

Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the tryptic-sensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Tryptic digestion of the enzyme complexed with the reaction-intermediate analogue released only two peptides by hydrolysis at Lys-8 and at Lys-14. The loss of susceptibility of Lys-466 to trypsin may be the result of a conformational change that limits the accessibility of the carboxyl-terminal region after binding of the reaction-intermediate analogue. Analysis of the amino-terminal tryptic peptide by composition and fast atom bombardment mass spectrometry demonstrated that the actual amino-terminal residue is proline at position 3 of the DNA-deduced sequence and that this proline is blocked with an N-acetyl moiety. Thus, posttranslational processing of the chloroplast-encoded large subunit of the enzyme must occur to remove Met-1 and Ser-2 and to acetylate the amino terminus.
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PMID:Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline. 342 48

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase. 838 54

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l-lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.
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PMID:In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor. 1666 Oct 24

The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 37 degrees C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Western blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 4 degrees C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 37 degrees C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation.
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PMID:Characteristics of ribulose-1,5-bisphosphate carboxylase/oxygenase degradation by lysates of mechanically isolated chloroplasts from wheat leaves. 1666 92

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.
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PMID:An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants. 1714 41

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO2/Mg(2+) -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO2/O2 specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K m values for RuBP were not significantly changed for the mutant enzymes, but the V max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO2/O2 specificity of Rubisco.
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PMID:Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii. 2419 54