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At least 667 records · Page 37Linked to original sources
PubMed1995

From growth to autolysis: the murein hydrolases in Escherichia coli.

Murein hydrolases cleave bonds in the bacterial exoskeleton, the murein (peptidoglycan) sacculus, a covalently closed bag-shaped polymer made of glycan strands that are crosslinked by peptides. During growth and division of a bacterial cell, these enzymes are involved in the controlled metabolism of the murein sacculus. Murein hydrolases are believed to function as pacemaker enzymes for the enlargement of the murein sacculus since opening of bonds in the murein net is needed to allow the insertion of new subunits into the sacculus. Furthermore, they are responsible for splitting the septum during cell division. The murein turnover products that are released during growth are further degraded by these (1 --> 6)-anhydromuramic acid derivatives by an intramolecular transglycosylation reaction.

Bacteriolysis↗
PubMed1981

Endogenous lipolytic activities during autolysis of highly enriched hepatic lysosomes.

High enriched (50- to 70-fold) fractions of "native" lysosomes were isolated using continuous flow electrophoresis from livers of rats which had not been pretreated with Triton WR-1339. Incubation of lysosomes for 30 min at pH 5.0 in the presence of 5 mM EDTA resulted in a dramatic loss in the content of fatty acids bound to triacylglycerols (137 down to 10 mumol/mg protein) and to phospholipids and an elevation in the level of unesterified fatty acid. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin concentrations decreased whereas those of lysophosphatidylethanolamine (0.8 up to 8.5% of total lipid-P) and lysophosphatidylcholine (1.9 up to 16.7%) rose in a manner parallel to their respective, fully acylated lipids. Other phospholipids, including phosphatidylinositol, did not change in concentration during incubation. These results indicate that lysosomal phospholipase A, sphingomyelin and triacylglycerol lipase are activated by incubation at acid pH, enabling them to hydrolyze endogenous lysosomal lipids. However, lysosomal phosphatidylinositol-directed phospholipase C is apparently unable to interact with phosphatidylinositol of the lysosomal membrane.

Animals↗
PubMed1989

Generation of a subunit III-like protein by autolysis of human and porcine proproteinase e in a binary complex with procarboxypeptidase A.

Tryptic treatment of human and porcine proproteinase E, procarboxypeptidase A binary complexes gave rise to active proteinase E after removal of an 11-residue N-terminal activation peptide. By contrast, upon treatment of either complex with active proteinase E, not only was the activation peptide released but also the hydrophobic dipeptide Val12-Val13 of the corresponding enzyme. No serine protease activity on specific synthetic peptide substrates could be detected. The structural homology of inactive proteinase E with subunit III of ruminant procarboxypeptidase A was strengthened by the existence of a functional homology since truncated proteinase E still possessed a weakly functional active site. Thus, subunit III-like proteins are generated by proteinase E-catalyzed limited proteolysis of proproteinase E.

Amino Acid Sequence↗
PubMed1984

Mechanism of postmortem autolysis of skeletal muscle.

Male Wistar rats were treated with the carboxyl, thiol, and serine protease inhibitors, pepstatin, Ep-475[L-trans-epoxysuccinyl-leucylamide(3-methyl) butane; E-64-c], and chymostatin. Then the femoral muscles of these rats and control animals were used for preparation of myofibril proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the degradation of these myofibril proteins with time (day) after death. The protease activities of the muscle were also measured. Tropomyosin was degraded most rapidly, followed by the heavy chain of myosin, alpha-actinin, and light chains of myosin (L1 and L2). Actin and troponin-T were degraded slowly, still remaining unchanged 2 weeks after death. The degradation of protein was not inhibited by pepstatin but was inhibited strongly by Ep-475 and very strongly by chymostatin. Chymostatin inhibited degradation of all components except alpha-actinin more strongly than Ep-475. Data on enzyme activities were consistent with these findings. These results suggest that after death the components of myofibrils are degraded with various proteases at various rates depending on their properties or their structure and that the proteases involved in the degradation show some specificity.

Animals↗
PubMed1996

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization.

The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to microM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca(2+)-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

Calcium↗
PubMed1988

Factors affecting the loss of mitochondrial function during zero-flow ischemia (autolysis) in slow and fast heart-rate hearts.

The (uninhibited) mitochondrial ATPase comprises approximately 90% of the total ATP hydrolyzing activity present in quiescent, ischemic canine heart muscle and its inhibition by its natural inhibitor protein plays a pivotal role in the slowing of tissue ATP depletion during ischemia. While dog heart mitochondria contain a full complement of mitochondrial ATPase inhibitor capable of fully down-regulating the enzyme activity present in this species, rat heart mitochondria contain a much lower level of inhibitor, sufficient to inhibit the enzyme activity present in this species by only approximately 20%. Moreover, this fractional complement of inhibitor remains largely inoperative in the ischemic rat heart. As shown in the present study, one apparent result of the lack of a functional complement of mitochondrial ATPase inhibitor in the rat heart is a more rapid rate of cell ATP depletion during zero-flow ischemia. This in turn results in a more rapidly developed and initially more severe cell acidosis in the ischemic rat heart because ATP hydrolysis produces protons. Finally, and consistent with earlier studies by us, the more rapid ATP depletion together with the more severe acidosis appears to result in a marked increase in the rate of loss of mitochondrial respiratory function in the ischemic rat heart compared to the ischemic dog heart. Our findings suggest that slow heart-rate hearts which contain in situ functional mitochondrial ATPase inhibitor, possess an effective mechanism for sparing cell ATP stores during early ischemia, whereas fast heart-rate hearts which lack in situ mitochondrial ATPase inhibitor function, possess a less effective ATP sparing mechanism.

Adenosine Triphosphatases↗
PubMed1995

Factors affecting autolysis of a subtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site.

The extracellular serine proteinase secreted by Ophiostoma piceae was degraded by autoproteolysis under certain conditions. At elevated temperatures the mature protein of 33 kDa was rapidly degraded without any accumulation of protein breakdown products. Glycerol, calcium ions and ammonium sulfate raised the heat stability of the enzyme, increasing its half-life at 45 degrees C from 1.9 min to 9.4 min, 40.4 min and 2 h, respectively. Thermal unfolding of the proteinase also occurred at higher temperatures in the presence of calcium ions and ammonium sulfate. Under conditions of heating, altered pH or partial depletion of protein-bound ions by EDTA, the structure of the proteinase was more susceptible to proteolysis. The major hydrolytic fragments of 19 kDa and 14 kDa, had N-terminal sequences of Ala1-Tyr2-Thr3-Thr4-Gln5-Thr6-Gly7-Ala8-Pro9-and Ser170-Glu171-Pro172-Se173-Val174-X-Thr 176-Val177-Gly178-Ala179-, respectively. Since the former sequence was identical to the N-terminus of the native protein, the major autoproteolytic cleavage site for a class II subtilase appeared to be the N-side of Ser170 using numbering based on the sequence of proteinase K. The secondary structure of the proteinase from O. piceae was similar to that of proteinase K based on circular dichroic spectra in the far ultraviolet region. This cleavage site was in a similar region to that identified for class I subtilases, which was located in an outer exposed loop of the tertiary structure of subtilisin-like serine proteinases.

Amino Acid Sequence↗
PubMed1989

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall.

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N''-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.

Aspergillus nidulans↗
PubMed1989

Beta-N-acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis.

A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.

Acetylglucosaminidase↗
PubMed2006

Following autolysis in proteases by NMR: insights into multiple unfolding pathways and mutational plasticities.

Biophysical studies in proteases are severely hampered due to the auto-cleavage property of these enzymes. In this context, we develop here a kinetic model and an NMR-based strategy to use this very autolytic property to derive useful insights into multiple unfolding pathways and mutational plasticities in these proteins. The basic idea lies in the interpretation of the auto-cleavage-driven decay of the folded protein peaks in the HSQC spectra as a function of time. The different peaks are seen to decay at different rates. As unfolding is the rate-determining step in the auto-cleavage reaction, the NMR spectral changes reflect on local unfolding processes at the residue level. A formalism is presented to gain insights into unfolding free energies and evaluate local perturbations due to single point mutations. The model is applied to HIV-1 protease-tethered dimer as an example, considering mutations at a particular site. Significant perturbations are seen even at very remote areas from the site of the mutation.

HIV Protease↗