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PubMed1994

Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase.

The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active-site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme-CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS-PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C-terminal fragment is blocked at its N-terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N-terminus corresponding to the site of proteolysis and a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active-site structure and catalytic mechanism of CoA transferase.

Amino Acid Sequence↗

PubMed2000

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).

Blotting, Western↗

PubMed1998

Alcohol and the pancreas.

Alcoholic pancreatitis may be one of the most serious adverse consequences of alcohol abuse. Its diagnosis, as it has for many years, depends primarily on clinical acumen in interpreting properly the symptoms and signs of abdominal distress, buttressed by elevated pancreatic enzymes (amylase and lipase). More recently, the use of computerized tomography (CT) in selected situations has been both of confirmatory and prognostic value. Severity of abnormality by CT correlates reasonably well with a variety of clinical-laboratory clusters (APACHE system, Ranson's criteria, etc.) and aids in therapy. The pathogenesis of alcoholic pancreatitis is not fully defined. The ultimate picture is one of tissue autolysis by activated proteolytic enzymes. The triggers for such activation, however, are still not known. They are represented by three main theories: (1) large duct obstruction and/or increased permeability relative to pancreatic secretion, (2) small duct obstruction due to proteinaceous precipitates, and (3) a direct toxic-metabolic effect of ethanol on pancreatic acinar cells. While not mutually exclusive, we favor the last hypothesis as being most consistent with the effects of ethanol on other organ systems. The direct effects of ethanol and/or its metabolites may be mediated, at least in part, via oxidative stress or the generation of fatty acid ethyl esters. Autolysis (regardless of proximate mechanism(s)) leads to inflammation likely mediated via release of various cytokines. It also should be appreciated that "acute" pancreatitis (the topic of this chapter) likely represents an acute process within a chronic pancreatic exposure and injury from alcoholic abuse. The key question of why pancreatitis develops in only a small number of alcohol abusers is not resolved. Therapy depends on the severity of alcoholic pancreatitis, which is defined by clinical-laboratory and often CT criteria. Mild pancreatitis usually resolves acutely with alcohol abstention and supportive therapy. Severe pancreatitis has a significant morbidity and mortality, mainly related to the degree of pancreatic necrosis and infection. It requires meticulous combined medical-surgical care.

APACHE↗

PubMed1979

The primary structure of porcine glandular kallikreins.

The amino acid sequence of the A- and B-chains of porcine pancreatic kallikrein B is presented and compared to that of porcine trypsin. The overall homology between both enzymes is 37% identical residues in corresponding position and 51% chemically similar resideus. Comparison of the sequences with the crystal structure of bovine trypsin reveals that the trypsin "autolysis loop" is enlarged in kallikrein by two residues but lacks the basic residue at the cleavage site. Substitutions at the calcium-binding site of trypsin which include Arg 70 for Glu 70 possibly interfere with ion binding. Insertions between trypsin residues 95 and 96 obviously form a new kallikrein "autolysis loop" containing the site of cleavage between the A- and B-chains. One carbohydrate moiety is attached to this surface loop at Asn 95, the second to Asn 239 at the same edge of the globular molecule. The residues at the surface of the substrate binding site are substituted to an extent of 85% while the residues forming contacts to the trypsin inhibitor (Kunitz) are highly preserved. Immunodiffusion studies as well as identity of the N-terminal sequences of pancreatic, submandibular and urinary kallikrein reveal the same genetic origin of the three glandular kallikreins.

Amino Acid Sequence↗

PubMed1990

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus.

The isolated cell wall of Streptomyces griseus 52-1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.

Amidohydrolases↗

PubMed1975

Cytochemical establishment of acid phosphatase in the bovine endometrium and trophoblast during implantation.

Acid phosphatase in the endometrial surface epithelium is seen in connection with autophagy and autolysis. In the precontact and initial apposition stage, enzyme-positive Golgi vesicles, lysosomes and secretion granules all indicate autophage performance of the dark uterine epithelial cells in the sense of a histiogene embryotrophe development. At the time of progressing apposition this is joined by cell degradation with the aim of histiolytical uterine milk production. Following the completed implantation in the adhesion phase no activity with autophagy and autolysis-correlated acid phosphatase can be established. In trophoblast giant cells the localisation of acid phosphatase speaks for secretional processes. The incidence of this enzyme in the adhesion stage in "ordinary" trophoblast cells leads to the supposition of autophage processes which must be investigated in more detail. The endometrial gland epithelium shows the same acid phosphatase-dependent autophage indications in the upper third of the glands as shown in the surface epithelium prior to apposition. However, the acid phosphatase activity and the secretion deduced therefrom, thus the histiogene embryotrophe development, is conserved during the whole early gravidity of the cow, independent of the implantation process.

Acid Phosphatase↗

PubMed1980

Ultraviolet induced lysosome activity in corneal epithelium.

A 5,000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm-2 to 10,000 Jm-2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hc) was 200 Jm-2 and lens threshold (HL) was 7,500 Jm-2. The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared.

Animals↗

PubMed1994

Susceptibility of C5b-9(m) to postmortem changes.

C5b-9(m) is a specific and sensitive marker for myocardial cell necrosis. The diagnostic value of this marker would be considerably limited in forensic practice if its immuno-histochemical demonstration were hampered by putrefaction or autolysis. We could demonstrate C5b-9(m) immunohistochemically in necrotic myocardium due to infarction up to the 11th day of experimentally induced putrefaction and autolysis, when reliable demonstration of myocardial infarction with hematoxylin-eosin was no longer possible. Under the experimental conditions of this study, no false positive immunohistochemical staining occurred.

Bias↗

PubMed1983

[Initial results of postmortem diagnosis of diabetes mellitus by Hb A1 determination].

Ensuring the diagnosis of diabetes mellitus by morphological findings is very difficult. Postmortem determination of blood glucose is of no value because of the influence of glycolysis. In clinical studies, Hb A1 determination is used for long-time therapy control of diabetes. Values of less than 10% Hb A1 show that assimilation of glucose is in order. This study is based on the investigation of blood from 174 cadavers (125 men aged between 17 and 84 years; 49 women aged between 27 and 89 years) with many different causes of death, including some cases of coma diabeticum. Blood was frozen immediately; in 48 cases it was stored at +4 degrees C, too. Besides, we took cerebrospinal fluid and urine from each corpse (frozen at -80 degrees C) for determination of glucose, lactic acid, and acetone. Hemoglobin A1 was analyzed by a chromatographic method: the concentrations of Hb A1 ranged from 7.5% to 20% independent of the actual amount of total hemoglobin in our samples (total Hb from 5 to 32 g/dl). Obviously, there is very little influence of autolysis or prefinal glucose fluctuations in blood on its quantity. There is a positive correlation to the concentration of glucose and lactic acid in cerebrospinal fluid (according to the formula of Traub) and also to the presence of acetone in case of diabetic coma. According to our experience, Hemoglobin A1 is a very useful marker to ensure the diagnosis of diabetes mellitus post mortem. As Hb A1 seems to be very constant and stable vs. autolysis, it may help to clear up cases of unexpected death.

Adolescent↗

PubMed1993

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases.

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new techniques have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Amino Acid Sequence↗

PubMed2006

A chitinase gene, chiB, involved in the autolytic process of Aspergillus nidulans.

Chitinases are thought to be involved in the morphogenesis and autolysis of filamentous fungi. We cloned a gene (chiB) encoding a class V chitinase from Aspergillus nidulans. ChiB expressed in Escherichia coli had chitin-hydrolyzing activity, indicating that chiB encoded a chitinase. Deletion of chiB affected neither germination efficiency nor hyphal growth rate, but considerably reduced the intracellular and extracellular chitinase activities. The decrease in hyphal dry weight during autolytic phase was slower in the mutant than in the wild-type strain. Western blot analysis indicated that the quantity of ChiB significantly increased when the wild-type mycelia were starved for carbon sources, a condition that induced hyphal autolysis. These results suggest that chiB plays an important role in the autolytic process in A. nidulans.

Amino Acid Sequence↗

PubMed2002

Milli-calpain from sea bass (Dicentrarchus labrax) white muscle: purification, characterization of its activity and activation in vitro.

A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24 degrees C in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required.

Journal Article↗

PubMed1976

On the subunit structure of particulate aminopeptidase from pig kidney.

Solubilization of particulate aminopeptidase (EC 3.4.11.2) from pig kidney with Triton X-100 yields an aggregate (mol. wt. approx. 10(6)) that decomposes into "free" aminopeptidase (mol. wt. 280 000) either upon autolysis at pH 5 or after exposure to trypsin. Both procedures yield free enzymes that are identical with respect to electrophoretic mobility, enzymatic activity and zinc content. After dissociation, the enzyme resulting from autolysis yields a single subunit of 140 000 molecular weight while the trypsin-treated enzyme produces three fragments (140 000, 95 000 and 48 000 mol. wt.). As the aggregate is formed by subunits 10 000 daltons heavier than those of the free enzyme, the existence of a hydrophobic portion anchoring the enzyme to the membrane might be postulated. Reactivation experiments carried out on the three purified fragments of urea-denatured aminopeptidase show that the 140 000 molecular weight subunit is the only one able to yield an active enzyme (after spontaneous dimerization). It can be concluded that the smaller fragments are artefacts resulting from trypsin degradation during purification.

Aminopeptidases↗

PubMed1977

Stabilization of bovine trypsin by reductive methylation.

Reductive methylation has little or no detectable effect on the catalytic or physicochemical properties of bovine trypsin but reduces its susceptibility to autolysis. Increased stability after methylation appears to result from the conversion of trypsin-susceptible lysine residues to trypsin-resistant epsilon-N,N-dimethyllysine residues. Reductively methylated trypsin is easily prepared and may be useful in place of trypsin where autolysis is otherwise difficult to control.

Animals↗

PubMed1984

Pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase and its isoenzymes in human myocardial biopsies and autopsies.

The extent to which pyridoxal-5'-phosphate stimulates the activities of aspartate aminotransferase and its cytoplasmic and mitochondrial isoenzymes was measured in six human left ventricular biopsies obtained freshly during open-heart surgery, and in 13 human left ventricular autopsies. A concentration of pyridoxal-5'-phosphate of 15 mumol/l for 1 h is sufficient to convert any apoenzyme to holo-enzyme. Pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase is 26 +/- 6% (+/- SD) in myocardial biopsies (range 22-34%) and 112 +/- 54% in myocardial autopsies (range 35-200%). The extent of stimulation of the cytoplasmic and mitochondrial isoenzymes is 18 +/- 9% and 32 +/- 6%, respectively, in myocardial biopsies, and 150 +/- 57% and 100 +/- 62%, respectively, in myocardial autopsies. The greater extent and variation of the pyridoxal-5'-phosphate-induced stimulation of aspartate aminotransferase and its isoenzymes in myocardial autopsies compared to that in myocardial biopsies is caused by autolysis and its duration. Autolysis depresses myocardial aspartate aminotransferase activity measured in the absence of pyridoxal-5'-phosphate which effect is more prominent for the cytoplasmic than for the mitochondrial isoenzyme.

Adult↗

PubMed1984

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing.

Canine prostate fluids and seminal plasma contain a major androgen-dependent protein which was identified as a proteolytic enzyme exhibiting an Arg-esterase activity. This protease, as characterized, is shown to be present as a two-chain structure held together by at least one disulfide bridge and composed of approximately 220 amino acids. Amino acid sequence determination of both chains has revealed a clear homology to other known amino acid sequences of serine proteases. Furthermore, the comparison of the presented 58 amino acids of the Arg-esterase with the other sequences revealed a very strong homology (larger than 50%) to members of the kallikrein family. The two chain structure could thus result from autolysis of a single chain enzyme in the 'kallikrein autolysis loop'. Amino acid composition of the canine prostatic enzyme suggests that it is related, but not identical, to pancreatic canine kallikrein.

Amino Acid Sequence↗

PubMed1986

Detection of antibodies to proteases used in laundry detergents by the radioallergosorbent test.

Two proteases, Esperase and Alcalase, derived from Bacillus licheniformis and B. subtilis, respectively, are used in laundry products. In testing for the prevalence of IgE antibodies to these enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these antigens. The first problem was the propensity of the allergen, Esperase, to undergo autolysis, suggesting that solid-phase Esperase might also lose reactivity through degradation. Treatment of Esperase with phenylmethylsulfonyl fluoride stabilized the enzyme and permitted the synthesis of a stable solid-phase antigen. The second problem was the finding that sera reactive with Esperase in the RAST were also reactive with Savinase, an enzyme from B. licheniformis to which the workers were not exposed. Immunochemical analyses of the three enzymes with specific rabbit antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent. RAST inhibition demonstrated that solid-phase Esperase possessed unique allergenic determinants in that the reactivity of IgE antibodies was inhibited by low concentrations of Esperase and only by very high concentrations of Alcalase and Savinase. In contrast, the reactivity of solid-phase Alcalase was occasionally inhibited equally well by Esperase and Alcalase. Most strikingly, the reaction of IgE antibodies with solid-phase Savinase was always inhibited by comparable quantities of Esperase, Alcalase, and Savinase. Thus, the establishment of the RAST for these proteases appears to require the use of phenylmethylsulfonyl fluoride to retard autolysis, and the results must be interpreted with caution because IgE antibodies in certain sera demonstrate cross-reactivity with Alcalase and Savinase.

Antibodies↗

PubMed1994

Infrared spectroscopic studies of calcium binding to inhibited beta-trypsins.

Fourier transform infrared spectroscopy was used to examine the effect of calcium binding on the secondary structure of two inhibited bovine beta-trypsins. Neither the diisopropyl fluorophosphate- nor benzamidine-inhibited forms showed detectable secondary structure perturbation upon calcium binding at pD 6.9 and 5.0, respectively. Considered in light of the recent assignment of an amide I' band to the autolysis loop of bovine beta-trypsin, these results contradict the generally held hypothesis that calcium slows trypsin autolysis by induction of a conformational change at this site and support the recent contention that the mechanism of action has a specific electrostatic origin. In addition, the appearance of a band at 1699 cm-1 in the benzamidine-inhibited form can be interpreted as resulting from the NC-N stretching vibrations of the amidinium moiety, which the observed crystal structure indicates is hydrogen-bonded to the carboxyl group of active-site Asp-189.

Animals↗