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PubMed1993

Membrane protein topology determination by proteolysis of maltose binding protein fusions.

A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest. Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain. A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm. A cytosolic MBP domain (MBP delta 2-26) is attached to the protein when its N-terminus resides in the cytoplasm. The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop. Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP. The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments. Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain. The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments. Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them.

ATP-Binding Cassette Transporters↗
PubMed1999

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography.

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.

Amino Acids↗
PubMed2001

Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases.

Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.

Colorimetry↗
PubMed2001

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F.

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.

Botulinum Toxins↗
PubMed1982

Bioorganic modelling stereoselective reactions with chiral neutral ligand complexes as model systems for enzyme catalysis.

In terms of the reporting of accomplished chemistry this review can do no more than give an indication of the rapid progress in the branch of bioorganic modelling based on the use of macrocyclic compounds that (usually) act as complexing agents. What remains to be done, however, is to point out problems that have not been satisfactorily solved and to suggest other profitable areas of investigation. From the material accumulated in this review one can draw the conclusion that especially crown (or cryptate) systems offer special advantages in bioorganic modelling because such compounds can - enzyme like - complex a potential substrate. On the basis of quite simple binding considerations, coupled with an analysis of steric interactions, accurate predictions of the stereochemistry of the complex can be made. The inclusion of catalytic groups in the crown (or cryptate) system and reactive functional groups in the substrate is then done in such a fashion that the stereoelectronic arrangement is compatible with the predicted geometry of the complex. However, the good complexing ability of the ligand is paradoxically often its greatest failing in terms of developing a system in which the functionalized ligand acts truly as a catalyst. As seen from much of the chemistry discussed in this review the ligand is incapable of the double task of complexing substrate but releasing product in an enzymic fashion, i.e. that turnover occurs. How is this problem to be solved? Induced conformational changes are an obvious approach although the design of proper systems remains a challenge for which few suggestions outside of unlimited ingenuity can be given. Much of the solution to such problems will lie also in a much better understanding than we now have of non-covalent interactions and the stereochemistry of such interactions. The assembly and disassembly of large molecular aggregates by the making and dissolution of non-covalent bonds is an art at which chemists are still relative amateurs. A better understanding of non-covalent interactions may also provide the key to achieving also the twin goals of both speed and selectivity in bioorganic modelling. As far as enantioselectivity is concerned it is clear that this can be achieved fairly effectively by the use of relatively small, but appropriately placed, groups that force the substrate to complex in an enantioselective step with the ligand. In other words, the problem of enantioselectivity can be solved at the stage of complex forming, which is kinetically rapid. The p]roblem of rate enhancement lies in the mentarity with the transition state of the reaction being catalyzed. Again the achievement of this goal lies in ingenuity of design. Potential areas of applications of chiral crown ether (or cryptate) ligand systems in bioorganic modelling lie in, for example, the formation of carbon-carbon bonds, development of oxidative processes (i.e...

Chemical Phenomena↗
PubMed2005

The ins and outs of intracellular peptides and antigen presentation by MHC class I molecules.

MHC class I molecules present small intracellular generated fragments to the outside surveying immune system. This is the result of a series of biochemical processes involving biosynthesis, degradation, translocation, intracellular transport, diffusion, and many more. Critical intermediates and end products of this cascade of events are peptides. The peptides are generated by the proteasome, degraded by peptidases unless transported into the ER where another peptidase and MHC class I molecules are waiting. Unless peptides bind to MHC class I molecules, they are released from the ER and enter the cytoplasm by a system resembling the ERAD pathway in many aspects. The cycle of peptides over the ER membrane with the proteasome at the input site and peptidases or MHC class I molecules on the output site are central in the MHC class I antigen presentation pathway and this review.

Animals↗
PubMed1984

Recent advances in protease research using synthetic substrates.

2-Naphthylamine, 4-methoxy-2-naphthylamine, 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin and X peptides have become a useful tool to study proteases by a battery of complementary methods; they now include biochemistry, section biochemistry, location cytochemistry, ultracytochemistry and isoelectric focusing. By the use of these procedures we hope that more insight will be possible in the functional role of proteases in health and disease in the near future.

Animals↗
PubMed1975

Peptide transport.

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Amino Acid Sequence↗
PubMed1986

The action of peptides and proteases on the arachidonate cascade of human and rat platelets.

The arachidonate cascade of human or rat platelets were found to be modified by peptides (bradykinin, angiotensin I, angiotensin II, Asp1-Val5-angiotensin II-amide, somatostatin) and proteases (trypsin, kallikrein). The lipoxygenase pathway was not altered by angiotensin I, angiotensin II, trypsin and kallikrein, while the synthesis some of the cyclooxygenase products was selectively changed by these substances. Bradykinin and somatostatin resulted in an attenuated formation of 12-HPETE and 12-HETE - U shape dose response curve, at the same time the synthesis of cyclooxygenase metabolites was increased - bell shape dose response curve. Asp1-Val5-angiotensin II-amide increased the synthesis of lipoxygenase products and diminished the formation of TxB2. At the same time this peptide selectively induced the enzymatic release of PGD2 from platelets. These peptides and proteolytic enzymes might have physiologic significance in the "Ying-Yang" balance in one hand between lipoxygenase and cyclooxygenase metabolites and on the other between the proaggregatory and antiaggregatory substances released from platelets.

Angiotensins↗
PubMed2000

Ladder sequencing.

Ladder sequencing of polypeptides involves progressive N- or C-terminal amino acid truncation via chemical or enzymatic treatments. Peptide ladders are generated in which each component differs from the next by one residue. The ladder components are analyzed by mass spectrometry, and the amino acid sequence is deduced from the mass differences between consecutive fragments. Chemical procedures are common in N-terminal degradation, whereas proteolytic digestion is often used in C-terminal sequence analysis. Matrix-assisted laser desorption/ionization mass spectrometry is widespread for one-step readout of the peptide ladders and provides high sensitivity in combination with robustness and ease of use. The particular advantage of ladder sequencing in relation to other techniques for sequence analysis is the high data acquisition rate and the very good sample throughput that can be achieved. Multiple determinations are carried out within minutes at high sensitivity and low sample consumption. Several reports demonstrate analysis at the low picomole to femtomole level.

Peptide Hydrolases↗
PubMed2002

Regulating the 26S proteasome.

Despite the fact that the composition of proteasomes purified from different species is almost identical, and the basic components of the proteasome are remarkably conserved among all eukaryotes, there are quite a few additional proteins that show up in certain purifications or in certain screens. There is increasing evidence that the proteasome is in fact a dynamic structure forming multiple interactions with transiently associated subunits and cellular factors that are necessary for functions such as cellular localization, presentation of substrates, substrate-specific interactions, or generation of varied products. Harnessing the eukaryotic proteasome to its defined regulatory roles has been achieved by a number of means: (a) increasing the complexity of the proteasome by gene duplication, and differentiation of members within each gene family (namely the CP and RPT subunits); (b) addition of regulatory particles, complexes, and factors that influence both what enters and what exits the proteasome; and (c) signal-dependent alterations in subunit composition (for example, the CP beta to beta i exchange). It is not be surprising that the proteasome plays diverse roles, and that its specific functions can be fine-tuned depending on biological context or need.

Adenosine Triphosphatases↗
PubMed1996

The proteolytic systems of lactic acid bacteria.

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

Amino Acid Sequence↗
PubMed1983

Proteolytic systems in lactic acid bacteria.

The proteolytic systems of lactic acid bacteria are important as a means of making protein and peptide N available for growth and as part of the curing or maturation processes which give foods their characteristic rheological and organoleptic properties. The proteolytic systems of lactic acid bacteria are described in relation to their growth and their functions in protein-rich foods. Their role in the manufacture of milk products is discussed.

Amino Acids↗
PubMed1979

Biochemical characterization of "LAP," a polymorphic aminopeptidase from the blue mussel, Mytilus edulis.

A genetically variable naphthylamidase enzyme, previously described as "leucine aminopeptidase," was purified approximately fiftyfold, and its biochemical properties were investigated. The enzyme was renamed "aminopeptidase I." Substrate affinities demonstrate that it is an alpha-aminoacyl peptide hydrolase (E.C. 3.4.11.-). Aminopeptidase I had a monomer molecular weight of 65--68,000, average of pI of pH 4.88, and broad pH optima between 6.5 and 8.0. The enzyme was inactivated rapidly between 40 and 50 C. Antibodies from purified enzyme did not cross-react with other naphthylamidases, but aminopeptidase I activity was inhibited by immune serum. The enzyme exhibited highest naphthylamidase activity for aromatic and hydrophobic aminoacyl naphthylamides. Aminopeptidase activity was highest for aromatic and hydrophobic N-terminal residues of tripeptides. Certain divalent metal cations, p-OH-mercuribenzoate, and N-ethylmaleimide were strongly inhibitory while chelating agents activated the enzyme.

Aminopeptidases↗
PubMed1984

Molecular weight heterogeneity of bovine serum transferrin.

Cattle transferrin (Tf) was purified from serum of variant A and four bands were isolated. The peptide patterns of these bands when cleaved by proteases and by cyanogen bromide (BrCN) were compared, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Variant A displays two groups of molecules--large (L) and small (S)--on SDS-PAGE; the molecular weight of the L bands is 78,400 +/- 1700 and that of the S bands is 72,000 +/- 1700. However, S-band molecules could not be produced artificially by heat treatment of L bands in the presence of SDS and 2-mercaptoethanol. Since deglycosylated Tf also showed molecular weight heterogeneity, the sugar moieties of Tf other than sialic acids were not the cause of the heterogeneity. These results suggest that heterogeneity within a given variant is due to the presence of two kinds of molecule of different molecular weight. The peptide patterns of L and S bands produced by proteases and those produced by BrCN were distinctly different from each other. However, the stepwise degradation patterns of L and S bands resembled each other when treated with both chymotrypsin and BrCN. This suggests that L-band molecules differ from S-band molecules only in the presence of an additional carboxyl-terminal peptide.

Alleles↗