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Biomedical subjects

M Orlowski

Publications and source records attributed to M Orlowski.

At least 37 records · Page 2Linked to original sources
PubMed1994

Proteolytic processing of farnesylated peptides: assay and partial purification from pig brain membranes of an endopeptidase which has the characteristics of E.C. 3.4.24.15.

The proteolytic processing of farnesylated peptides was investigated with the tritiated C-terminal heptapeptide of mouse N-Ras protein, Propionyl-Gly-Ser-Pro-(farnesyl-Cys)-[3H]Val-Leu-Met, as substrate. Using two rapid and sensitive methods, the peptidase which cleaves the (farnesyl-Cys)-[3H]Val bond was purified more than 100 times from a microsomal fraction of pig brain. The determination of its molecular mass (about 70 kDa) and its pH range for optimum activity (neutral pH) as well as the results obtained with various inhibitors indicate that this [3H]Val-Leu-Met releasing enzyme resembles endopeptidase 24.15: a thiol-dependent zinc metallopeptidase. Parallel and comparative inhibition assays on partially purified enzyme and pure "24.15" confirm this similarity.

Amino Acid Sequence↗
PubMed1994

Central administration of the endopeptidase 24.15 inhibitor cFP-AAF-pAB suggests dynorphin as the endogenous ligand underlying behavioral effects of milk in the fetal rat.

Intraoral infusion of milk to the rat fetus promotes opioid activity that results in reduced responsiveness in a behavioral bioassay involving perioral cutaneous stimulation. Intracisternal administration of cFP-AAF-pAB, an inhibitor of endopeptidase 24.15, prolonged the opioid activity induced by milk infusion. Treatment with the selective kappa opioid antagonist nor-binaltorphimine blocked the effect of cFP-AAF-pAB on milk-induced opioid activity, but treatment with the mu antagonist CTOP or the delta antagonist naltrindole did not. These findings imply that milk may exert its effect on fetal behavior by increasing levels of dynorphin in the fetal central nervous system.

Amino Acid Sequence↗
PubMed1994

Effects of a metalloendopeptidase-24.15. Inhibitor on renal hemodynamics and function in rats.

N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), an active-site-directed inhibitor of metalloendopeptidase-24.15, has been shown to lower blood pressure, increase cardiac output and renal blood flow, and potentiate the intravenous bradykinin-induced vasodepressor response. Because in vivo cFP-AAF-pAB can be converted to N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala (a compound with angiotensin converting enzyme inhibitory activity) by metalloendopeptidase-24.11, it is possible that some of its effects are due to angiotensin converting enzyme inhibition. In the present study, we questioned (1) whether cFP-AAF-pAB inhibits angiotensin converting enzyme in vivo and (2) whether cFP-AAF-pAB has renal effects that are independent of its conversion to an angiotensin converting enzyme inhibitor. cFP-AAF-pAB alone (3 mumol in 300 microL per rat) almost abolished the blood pressure response to angiotensin I, suggesting that in vivo it inhibits angiotensin converting enzyme. In rats pretreated with a high dose of enalaprilat (1 mg/kg), cFP-AAF-pAB had no further effect on blood pressure, renal blood flow, or potentiation of the vasodepressor response to bradykinin but still increased glomerular filtration rate by 44 +/- 9% (P < .01); urine volume increased by 118 +/- 10% (P < .001), urinary sodium excretion by 230 +/- 31% (P < .001), urinary potassium excretion by 68 +/- 14% (P < .01), and urinary cyclic GMP by 55 +/- 18% (P < .01). All of these changes were significant compared with enalaprilat/vehicle-treated rats. Fractional excretion of sodium and potassium did not differ from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed1993

Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids.

Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Mitochondrial genome of the dimorphic zygomycete Mucor racemosus.

Mitochondria were isolated from the dimorphic zygomycete Mucor racemosus by differential centrifugation. DNA from the organelles was purified by cesium chloride-ethidium bromide isopycnic centrifugation. Examination of the mitochondrial DNA by electron microscopy revealed a circular chromosome approximately 63.8 kbp in circumference. The chromosome was digested with restriction endonucleases and the resulting DNA fragments were separated by agarose-gel electrophoresis. Electophoretic mobilities and stoichiometry of the fragments indicated a mixed population of mtDNA molecules each with a size of about 63.4 kbp. Physical maps were constructed from analyses of fragments generated in single and double restriction digests and from the hybridization of fragments to probes for the large and small mitochondrial rRNA genes from Saccharomyces cerevisiae. The Mucor mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes.

Cloning, Molecular↗
PubMed1992

Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.

Amino Acid Sequence↗
PubMed1992

Immunocytochemical localization of endopeptidase 24.15 in rat brain.

Endopeptidase 24.15 (EC 3.4.24.15; EP 24.15), a zinc-metalloendopeptidase highly active in rat testes, brain and pituitary, converts some prodynorphin- and proenkephalin-derived oligopeptides into the corresponding enkephalins and degrades a variety of bioactive peptides including bradykinin, neurotensin, and both angiotensin I and II. The immunocytochemical localization of the enzyme was studied in rat brain using a polyclonal antibody raised in rabbits against a homogeneous preparation of the enzyme isolated from rat testes. The distribution of EP 24.15 immunoreactivity in the brain was widespread, being present in both neurons and glial cells. Surprisingly, however, staining was predominantly nuclear, and not cytoplasmic as expected based on the biochemical demonstration that EP 24.15 activity is predominantly associated with the soluble protein fraction of brain homogenates. Cytoplasmic staining was detected in large neurons but was less intense than the nuclear staining. The highest density of EP 24.15-staining was detected in nuclei of cerebellar Purkinje cells and in hippocampal dentate gyrus cells. High levels of immunoreactivity were also noted in brain areas which contain peptides known to be substrates of the enzyme in vitro. This localization supports a role for EP 24.15 in neuropeptide metabolism, but also suggests an as yet undefined role in nuclear function.

Animals↗
PubMed1992

Endopeptidase 24.15 inhibition and opioid antinociception.

Whereas endopeptidase 24.11 cleaves the Gly-Phe bond in both Met- and Leu-enkephalin, endopeptidase 24.15 rapidly converts dynorphin A1-8, alpha and beta-neoendorphin into Leu-enkephalin, and Met-enkephalin-Arg6-Gly7-Leu8 (MERGL) into Met-enkephalin. Inhibitors of both endopeptidase 24.11 and endopeptidase 24.15 each produce antinociception, and inhibitors of endopeptidase 24.11 increase the magnitude of enkephalin antinociception. The present study compared the central antinociceptive effect of an inhibitor of endopeptidase 24.15, N-[1-(R-S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) with one of endopeptidase 24.11 N-[1-(RS)-carboxy-3-phenylpropyl]-Phe-p-aminobenzoate (cFP-F-pAB) upon central opioid antinociception induced by MERGL, metenkephalin and dynorphin A1-8. cFP-AAF-pAB, but not cFP-F-pAB increased MERGL antinociception on the tail-flick and jump tests. In contrast, cFP-F-pAB, but not cFP-AAF-pAB increased met-enkephalin antinociception. Whereas central dynorphin A1-8 failed to induce antinociception itself, co-administration of cFP-AAF-pAB and dynorphin A1-8 increased nociceptive thresholds. This effect was not accompanied by motor dysfunction, but was blocked by systemic pretreatment with naloxone or central pretreatment with naltrexone or nor-binaltorphamine, but not beta-funaltrexamine. These data indicate that endopeptidase 24.15 may be responsible for the degradation of specific opioid peptides (e.g., MERGL, dynorphin), and that this process may prevent the full expression of their antinociceptive properties.

Amino Acid Sequence↗
PubMed1992

Effect of doxapram on neostigmine evoked antagonism of vecuronium neuromuscular block.

We have examined the effect of doxapram on neostigmine-evoked antagonism of vecuronium neuromuscular block in a double-blind study in anaesthetized patients. Neuromuscular transmission was measured with a Datex Relaxograph. The mean time to recovery of the first contraction of the train-of-four (T1) from 25% to 75% was significantly longer in the presence of doxapram (138 (SEM 21) s; n = 23) than in its absence (95 (13) s; n = 29) (P = 0.014). The recovery of the train-of-four ratio was prolonged also in the presence of doxapram, although this difference was not statistically significant.

Adult↗
PubMed1991

Regulation of the peptidylglutamyl-peptide hydrolyzing activity of the pituitary multicatalytic proteinase complex.

The finding that the activity of the multicatalytic proteinase complex (MPC) is greatly activated by low concentrations of sodium dodecyl sulfate (SDS) and fatty acids led to the proposal that the proteolytic activity of the complex is latent and that activation is needed for expression of full activity. Kinetic examination of the nature of the latency with Cbz-Leu-Leu-Glu-2-naphthylamide, a substrate cleaved by the peptidylglutamyl-peptide hydrolyzing activity (PGPH activity) of the complex, showed that plots of velocity versus substrate concentration yield sigmoidal curves, implying the presence of two or more substrate binding sites and the presence of cooperative interactions between the sites. Hill plots of log [v/(Vmax-v)] versus log [S] gave slopes with a Hill coefficient of 2.2-2.4, suggesting that more than two subunits are expressing the PGPH activity. At saturating substrate concentrations, SDS and lauric acid exposed a masked component of PGPH activity that was about equal in magnitude to the overt activity measured in the absence of these detergents, showing that under the latter conditions only about half of the enzyme activity is expressed. Activation by SDS and lauric acid was greater at low than at high substrate concentrations and was associated with a shift of the substrate concentration at half-Vmax (apparent Km) toward lower values. The decrease in the apparent Km in the presence of SDS (but not in the presence of lauric acid) was associated with a decrease in cooperativity. The presence of at least two distinct PGPH activity components with different reactivities was also indicated by the finding of two distinct inactivation rate constants in reactions with 3,4-dichloroisocoumarin, an irreversible inhibitor of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Increases in opioid-mediated swim antinociception following endopeptidase 24.15 inhibition.

The duration of action and potency of endogenous opioid peptides are limited by proteolytic enzymes such as endopeptidases 24.11 and 24.15. Whereas endopeptidase 24.11 cleaves enkephalin pentapeptides, endopeptidase 24.15 degrades longer-chained opioids including dynorphin A1-8 and met-enkephalin-Arg6-Gly7-Leu8 (MERGL). Inhibitors of endopeptidase 24.11 and 24.15 both increase basal nociceptive thresholds and respective forms of opioid antinociception. Acute exposure to certain environmental stressors can produce antinociception which is opioid mediated; inhibitors of endopeptidase 24.11 potentiate this effect. The present study evaluated whether central administration of a selective inhibitor of endopeptidase 24.15, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) increased antinociception following intermittent cold-water swims (ICWS) in rats. cFP-AAF-pAB (0.25-25 nmol, ICV) dose-dependently increased ICWS antinociception on the tail-flick and jump tests without affecting basal nociceptive thresholds. The opioid mediation of ICWS antinociception was confirmed by significant reductions in this response following naloxone. These data indicate that longer-chained endogenous opioid peptides participate in the antinociception induced by ICWS.

Animals↗
PubMed1991

Mucor dimorphism.

Mucor dimorphism has interested microbiologists since the time of Pasteur. When deprived of oxygen, these fungi grow as spherical, multipolar budding yeasts. In the presence of oxygen, they propagate as branching coenocytic hyphae. The ease with which these morphologies can be manipulated in the laboratory, the diverse array of morphopoietic agents available, and the alternative developmental fates that can be elicited from a single cell type (the sporangiospore) make Mucor spp. a highly propitious system in which to study eukaryotic cellular morphogenesis. The composition and organization of the cell wall differ greatly in Mucor yeasts and hyphae. The deposition of new wall polymers is isodiametric in yeasts and apically polarized in hyphae. Current research has focused on the identity and control of enzymes participating in wall synthesis. An understanding of how the chitosome interacts with appropriate effectors, specific enzymes, and the plasma membrane to assemble chitin-chitosan microfibrils and to deposit them at the proper sites on the cell exterior will be critical to elucidating dimorphism. Several biochemical and physiological parameters have been reported to fluctuate in a manner that correlates with Mucor morphogenesis. The literature describing these has been reviewed critically with the intent of distinguishing between causal and casual connections. The advancement of molecular genetics has afforded powerful new tools that researchers have begun to exploit in the study of Mucor dimorphism. Several genes, some encoding products known to correlate with development in Mucor spp. or other fungi, have been cloned, sequenced, and examined for transcriptional activity during morphogenesis. Most have appeared in multiple copies displaying independent transcriptional control. Selective translation of stored mRNA molecules occurs during sporangiospore germination. Many other correlates of Mucor morphogenesis, presently described but not yet explained, should prove amenable to analysis by the emerging molecular technology.

Carbon↗
PubMed1990

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15.

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.

Amino Acid Sequence↗