Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

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.
...
PMID:A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides. 135 Jul 86

The mitochondrial import of a member of the "chaperonin" group of proteins which play an essential role in the import of protein into organelles and their subsequent proper folding has been examined. The cDNA for human hsp60 (synonyms: GroEL homolog, P1) was transcribed and translated in vitro and its import into isolated rat heart mitochondria examined. The protein was converted into a mature form of lower molecular mass (= 58 kDa) which was resistant to trypsin treatment. The import of human hsp60 into mitochondria was inhibited in the presence of an uncoupler and also no import occurred when the N-terminal presequence was lacking. These results indicate that the chaperonin protein(s) are transported into mitochondria by a process similar to other imported mitochondrial proteins. Our results also indicate that although the P1 protein precursor was efficiently imported into mitochondria, in comparison to precursors of other mitochondrial proteins (viz. ornithine carbamoyltransferase and uncoupling protein) much less binding of pre P1 to mitochondria was observed. The significance of this latter observation at present is unclear.
...
PMID:Mitochondrial import of the human chaperonin (HSP60) protein. 197 19

The urea denaturation of the chaperonin GroEL has been studied by circular dichroism, intrinsic tyrosine fluorescence and fluorescence of the hydrophobic probe, 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). It is shown that GroEL denaturation, monitored by CD and intrinsic fluorescence measurements, can be well described by a two-state transition that is complete by 3-3.1 M urea. The beginning of this transition overlaps the urea concentrations where the oligomeric protein starts to dissociate into individual monomers. Subsequent addition of the denaturant leads to complete unfolding of the monomers. Monomers unfolded at urea concentrations higher than 3.1 M are not competent to form their native conformations under the conditions employed here, and they are not able to reassemble to oligomers upon dilution of urea. In contrast to the CD and intrinsic fluorescence measurements, bisANS bound to GroEL exhibits considerable fluorescence intensity under conditions where the CD and intrinsic fluorescence signals have already reached their minimum values (> 3.1 M urea). This binding of bisANS, under conditions where the majority of the secondary structure of GroEL has already unfolded, indicates the existence of hydrophobic residual structure. This structure cannot be detected by CD measurements, but it can be unfolded by raising further the urea concentration. The existence of this structure does not depend on the source or method of the protein preparation. Intrinsic fluorescence and trypsin digestion demonstrate no difference between the bisANS-bound form of GroEL and the free form of the protein, showing that the GroEL structure is not greatly affected by the interaction with bisANS.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Residual structure in urea-denatured chaperonin GroEL. 757 88

The use of diode-array detection allows the non-destructive identification of tryptophan and tyrosine residues in complex peptide mixtures separated by capillary electrophoresis. Second-order derivative spectra of both amino acids show significant differences while zero-order spectra are overlapping to a great extent. A mixture of peptides containing tryptophan and/or tyrosine residues was used to evaluate this method. Tryptic peptide maps of carbonic anhydrase and of the bacterial chaperonin protein GroEL, and of an autodigest of trypsin were characterized for tryptophan- and tyrosine-containing peptides. Automated spectra library search was performed successfully.
...
PMID:Identification of tryptophan and tyrosine residues in peptides separated by capillary electrophoresis by their second-derivative spectra using diode-array detection. 795 88

Two site-directed mutants of the enzyme rhodanese which replace glutamic acid 17 with either glutamine (E17Q) or with proline (E17P) were produced and purified. Both mutants displayed specific activities similar to the wild type enzyme. E17Q was equivalent to the wild type enzyme in all assayed characteristics, except that the mutant had slightly more solvent exposure of hydrophobic surfaces. Results with E17Q suggest that the charge on Glu17 is not required for helix stabilization, nor is its titration required for the low pH structural transitions seen previously. In contrast, E17P was significantly different from the wild type enzyme. For example, E17P had (a) higher exposure of hydrophobic surfaces in the unperturbed state; (b) considerably lower stability to perturbation by urea; (c) easier exposure of organized hydrophobic surfaces on initial unfolding, even though denaturation to the final disorganized state was the same as for the wild type; (d) the ability to refold without assistants but with lower yields and somewhat slower folding; and (e) similar susceptibility to trypsin and evidence of a new clip site closer to the NH2 terminus. However, E17P and the wild type enzyme had very similar recoveries with chaperonin-assisted refolding, and the chaperonin protein groEL had a very similar ability to suppress unassisted refolding. These results indicate that changes in the NH2-terminal sequence can have dramatic effects on the stability of rhodanese and on its ability to be refolded in the absence of assistants. They further suggest that interactions with chaperonins do not rely exclusively on the detailed conformation at the NH2 terminus. A model that incorporates observations here includes step(s) in which the NH2-terminal sequence folds onto the NH2-terminal domain late in the folding process after the protein had adopted a near native conformation.
...
PMID:The folding and stability of rhodanese are influenced by the replacement of glutamic acid 17 in the NH2-terminal helix by proline but not by glutamine. 809 37

E. coli GroEL chaperonin monomers, isolated after urea-induced dissociation of GroEL14, undergo cold denaturation below 5 degrees C. Above 5 degrees C, these monomers undergo MgATP-dependent self-assembly. We have demonstrated a conformational transition at 0 degree C induced by interaction of monomeric GroEL with adenine nucleotides. This conformation has a dramatically decreased Stokes radius and enhanced resistance to trypsin but it is slightly less compact than the conformation of monomers at 23 degrees C in the absence of MgATP and it is not capable of spontaneous self-assembly. A second, temperature-dependent conformational change with a transition at about 5 degrees C is required for GroEL to undergo oligomerization.
...
PMID:Stabilization of a compact conformation of monomeric GroEL at low temperature by adenine nucleotides. 809 30

Although the role of nucleotides in the catalytic cycle of the GroESL chaperonin system has been extensively studied, the molecular effects of nucleotides in modulating exposure of sites on GroEL has not been thoroughly investigated. We report here that nucleotides (ATP, ADP, or adenosine 5'-(beta, gamma-imino)triphosphate) in the presence of Mg2+ make the oligomer selectively sensitive to trypsin proteolysis in a fashion suggesting conformational changes in the monomers of one heptameric ring. The site of proteolysis in the monomer that is exposed upon nucleotide binding by the oligomer is in the apical domain (Arg-268). Further, complexes of GroEL with GroES or rhodanese display the same sensitivity to proteolysis, unlike the GroEL-GroES-rhodanese complex, which is protected from proteolysis. The influence of various cations on trypsin proteolysis is investigated to elucidate the differential effects that monovalent and divalent cations have on the oligomeric structure of the chaperonin. These results are discussed in relation to the molecular basis for the chaperonin activity.
...
PMID:Ligand-induced conformational changes in the apical domain of the chaperonin GroEL. 855 May 66

Ligand-induced conformational changes of GroEL alone and with bound rhodanese, citrate synthase, or dihydrofolate reductase were studied by limited proteolysis. Similar digestion patterns of GroEL, with or without bound substrate polypeptide, were obtained in the absence and presence of the chaperonin ligands, K+, Mg2+, or ATP. The rates of formation and degradation of the six produced proteolytic fragments were significantly different, however. Strikingly, only with Mg2+/ATP or K+/Mg2+/ATP an additional fragment of approximately 25 kDa was generated during digestion of GroEL alone or with bound rhodanese or dihydrofolate reductase, but not with bound citrate synthase. Most of the trypsin-sensitive sites in GroEL were localized in the flexible apical domain, which contains the putative polypeptide-binding region. Our data indicate that subtle structural changes in the trypsin-sensitive regions of GroEL occur as a result of the binding of the chaperonin ligands. However, these structural changes are influenced by the GroEL substrate polypeptides.
...
PMID:Ligand-induced conformational changes of GroEL are dependent on the bound substrate polypeptide. 866 87

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.
...
PMID:Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells. 926 Aug 87

A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo.
...
PMID:Novel 30 kDa protein possessing ATP-binding and chaperone activities. 929 Nov 33


1 2 3 Next >>

---