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Biomedical subjects

M Orlowski

Publications and source records attributed to M Orlowski.

At least 73 records · Page 4Linked to original sources
PubMed1986

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin.

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Increased cathepsin B-like activity in alveolar macrophages and bronchoalveolar lavage fluid from smokers.

Cathepsin B-like activity was determined in alveolar macrophages (AM) and cell-free bronchoalveolar lavage fluid (BALF) obtained from volunteers who were current cigarette smokers and compared with that found in lifetime nonsmokers. Enzyme activity was determined with benzyloxycarbonyl-Leu-Leu-Arg-2-naphthylamide as the substrate. Specific activity of the enzyme was more than twice as high in AM from smokers than in cells from nonsmokers (35,600 +/- 2,250 versus 16,000 +/- 860; p less than 0.001) and about 10 times as high in BALF from smokers than in that from nonsmokers (3,060 +/- 380 versus 300 +/- 25; p less than 0.001). Because cathepsin B is capable of degrading structural lung proteins and inactivating alpha-1-proteinase inhibitor, and the elastinolytic activity of AM may be mediated through cysteine proteinases such as cathepsin B, the finding of high concentrations of an enzyme with cathepsin B-like activity in AM and BALF from smokers suggests the need to explore the role of the enzyme in structural lung damage associated with cigarette smoking.

Adult↗
PubMed1985

Cathepsin B and D activity in stimulated peritoneal macrophages.

Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.

Animals↗
PubMed1985

Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides.

A soluble metalloendopeptidase identified in rat brain, preferentially cleaves bonds in peptides having hydrophobic amino acid residues in the P1, P2, and P3' positions. (The nomenclature of T. Schechter and A. Berger is used to describe the interaction between enzyme and substrate. The amino acid residues in the substrate are designated as P1, P2, P3 etc. in the N-terminal direction and P1', P2', P3' etc. in the C-terminal direction from the bond undergoing cleavage. The corresponding subsites in the enzyme are identified by the letter S.) The degradation of a series of biologically active peptides and their affinity toward the enzyme was studied. Dynorphin-(1-8), alpha-neo-endorphin, and beta-neo-endorphin are rapidly hydrolyzed to form leu-enkephalin, whereas bovine adrenal medulla dodecapeptide is hydrolyzed to form met-enkephalin. The enzyme, however, does not cleave a larger precursor molecule of metenkephalin, such as bovine adrenal medulla docosapeptide. Several other bioactive peptides are also cleaved at sites consistent with our previously reported specificity studies. Met- and leu-enkephalin are resistant to hydrolysis. The binding affinity [as expressed by inhibitory constant (Ki) or Michaelis-Menten constant (Km) values] of several bioactive peptides such as dynorphin-(1-8), beta-neo-endorphin, neurotensin, angiotensin I, and bradykinin was found to be in the micromolar range. These peptides were also rapidly hydrolyzed by the enzyme, showing, as a result, high specificity constants (kcat/Km values). The highest enzyme activity was found in brain, testis, and in the anterior and posterior lobes of the pituitary, while the activity in such tissues as spleen, liver, kidney, lung, adrenals, and thyroid amounted to only 10-20% of that found in brain. This distribution of enzyme activity, together with its preference for oligopeptides as substrates, its ability to generate leu- and met-enkephalin from several larger peptide precursors, and its affinity toward several other bioactive peptides, suggests that the enzyme functions in the metabolism of neuropeptides.

Animals↗
PubMed1984

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain.

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Inhibitors of an enkephalin degrading membrane-bound metalloendopeptidase: analgesic properties and effects on striatal enkephalin levels.

Intraperitoneal administration of N-[1-(R,S)-carboxy-2-phenylethyl-Phe-p-aminobenzoate, synthesized in this laboratory as a potent inhibitor of membrane-bound metalloendopeptidase (EC 3.4.24.11) caused a prolonged but weak analgesic effect on rats as measured by the tail flick test. It also caused a transitory but significant increase in striatal [Leu5]- and [Met5]enkephalin levels 3 h, after administration. Analogs of the inhibitor in which the phenylalanyl residue was replaced by an alanyl or glycyl residue also elicited prolonged analgesic responses although their inhibitory potencies were 75 and more than 1500 times lower respectively. The glycine containing derivative did not alter striatal enkephalin levels 3 h, after administration. The data suggest that inhibition of the metalloendopeptidase decreases the rate of degradation of endogenous enkephalins, however the analgesic properties of the inhibitors do not seem to be related to their inhibitory potencies. Factors other than changes in striatal enkephalin levels may contribute to the analgesic effect of the three N-carboxyphenylethyl derivatives.

4-Aminobenzoic Acid↗
PubMed1984

A sensitive procedure for determination of cathepsin D: activity in alveolar and peritoneal macrophages.

Several new synthetic substrates fulfilling the specificity requirements of cathepsin D were synthesized. One of these D-Phe-Ser(O-CH2-C6H5)-Phe-Phe-Ala-Ala-pAB(pAB = p-aminobenzoate) proved to be highly sensitive and convenient for measuring activity. Enzyme determination was carried out in a two-step reaction. In the first step the enzyme hydrolyzes the Phe-Phe bond of the substrate at pH 3.4. In the second step aminopeptidase M (EC 3.4.11.2) degrades one of the products Phe-Ala-Ala-pAB at pH 7 to 8 with the release of free pAB, which is then determined by a diazotization procedure. Activity can be measured in as little as 1 to 5 micrograms of macrophage protein. The activity of cathepsin D in rat alveolar macrophages was almost ten times higher than in resident peritoneal macrophages, and more than 25 times higher than in blood monocytes. The data indicate that transformation of blood monocytes into macrophages is associated with a much greater increase of cathepsin D activity in alveolar than peritoneal macrophages.

Aminopeptidases↗
PubMed1984

Biochemical and immunological properties of a membrane-bound brain metalloendopeptidase: comparison with thermolysin-like kidney neutral metalloendopeptidase.

Membrane-bound neutral metalloendopeptidase ("enkephalinase") was purified from rabbit brain and compared with a homogeneous preparation of a similar enzyme (EC 3.4.24.11) isolated from rabbit kidney. The two enzymes had the same pH optimum and the same apparent molecular weight. They showed identical specificity toward several synthetic substrates and cleaved both Met- and Leu-enkephalin at the Gly-Phe bond. Minor, but significant, differences were found between the two enzymes in the inhibitory constants determined for phosphoramidon and the N-[1(R,S)-carboxy-2-phenylethyl] derivatives of phenylalanyl and alanyl-p-aminobenzoate. A guinea pig antiserum obtained against the rabbit kidney enzyme showed strong crossreactivity with the rabbit brain enzyme when tested in an anticatalytic immunoinhibition assay. Ouchterlony immunodiffusion experiments gave a pattern of precipitation consistent with partial identity of the two enzymes. The kidney enzyme, however, seemed to contain antigenic determinants not present on the brain enzyme. The data indicate that the two enzymes are identical with respect to specificity, pH optimum, and molecular weight, but show minor, although significant, differences in interaction with active-site-directed inhibitors and specific antisera.

Animals↗
PubMed1984

Prolyl endopeptidase: inhibition in vivo by N-benzyloxycarbonyl-prolyl-prolinal.

The activity of prolyl endopeptidase in homogenates of mouse tissues was determined 30 min after intraperitoneal injection of N-benzyloxycarbonyl-prolyl-prolinal (1.25 mg/kg), a potent transition state analog inhibitor (K1 = 14 nM) of prolyl endopeptidase (EC 3.4.21.26). A more than 85% decrease of enzyme activity was obtained in all tissues. The in vivo degradation of potential prolyl endopeptidase substrates was studied by following the release of sulfamethoxazole from N-benzyloxycarbonylglycyl-prolyl-sulfamethoxazole, a model synthetic substrate of the enzyme. When this substrate was given intraperitoneally, its enzymatic degradation was blocked after administration of the inhibitor in a dose- and time-dependent manner, indicating inhibition of the enzyme in vivo. Of interest is the long duration of the inhibition. After a relatively low inhibitor dose (5 mg/kg) significant inhibition was seen in most tissues even after 6 h. The brain was particularly sensitive to the effect of the inhibitor. Since prolyl endopeptidase readily degrades many proline-containing neuropeptides, the inhibitor should be of value in studies on the role of the enzyme in neuropeptide metabolism.

Animals↗
PubMed1984

Differential gene expression during aerobic germination of Mucor racemosus sporangiospores.

Evidence is provided suggesting that several modes of differential gene expression operate concomitantly during the first 60 min of germination of Mucor racemosus sporangiospores under air. Protein synthesis was initiated immediately upon exposure of the spores to nutrient medium and accelerated exponentially throughout the period of observation. All translation during the first 30 min of germination occurred using only preformed stable mRNAs as a template. Analysis of the protein products synthesized in vivo was performed by L-[35S]methionine labeling, one- and two-dimensional polyacrylamide gel electrophoresis, and autoradiography. The population of proteins accumulated during spore formation and present at the time of harvest differed significantly from those proteins synthesized during spore germination. Autoradiographs displayed several proteins synthesized during the former but not the latter morphogenetic process. Conversely, other proteins were synthesized during the first 30 min of germination but not during spore formation, even though the mRNA specifying these proteins must have been synthesized and stored in the dormant spore. A posttranscriptional regulatory mechanism that directs selective translation thus appears to exist in the developing spore. In addition, autoradiographs showed that many proteins, although made throughout the intervals examined, displayed significant changes in their relative rates of synthesis. One gene product exemplified a possible case of post-translational modification during the first hour of sporangiospore germination.

Aerobiosis↗
PubMed1984

Peptide-degrading enzymatic activities in GH3 cells and rat anterior pituitary homogenates.

The activities of a number of peptide-degrading enzymes were compared in homogenates of GH3 cells and rat anterior pituitaries. The enzymes studied were prolyl endopeptidase (EC 3.4.21.26), a soluble metalloendopeptidase, pyroglutamyl peptide hydrolase (EC 3.4.11.8), a multicatalytic protease complex, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), aminopeptidase (EC 3.4.11.2), and a membrane-bound neutral metalloendopeptidase (EC 3.4.24.11). Specific substrates were used to measure the activities, and active-site-directed inhibitors were used to verify the identities of the enzymes studied. Of the two lysosomal enzymes studied, cathepsin B, the enzyme with the highest activity in both preparations, had 5 times the activity in GH3 cell homogenates as in anterior pituitary homogenates. Cathespin D had a somewhat higher activity in the anterior pituitary homogenates than in the GH3 cell homogenates. Soluble metalloendopeptidase and prolyl endopeptidase, both cytoplasmic enzymes, had about twice the activity in GH3 cell homogenates as in anterior pituitary homogenates. Membrane-bound neutral metalloendopeptidase in the GH3 cell homogenates had 25% of the activity of the anterior pituitary homogenates. Of the two TRH-degrading enzymes, the activity of prolyl endopeptidase in GH3 cell homogenates was about 25 times higher than that of pyroglutamyl peptide hydrolase. Since the secretory function of the pituitary is in part controlled by neuropeptides, the knowledge of the enzyme profiles of the GH3 cells and the anterior pituitary should be of value in studying the metabolism of neuropeptides and peptide hormones in these systems.

Animals↗