Sherwin Wilk

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.

Abstract: Pituitary cation‐sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin‐like activity), basic amino acids (trypsin‐like activity), and acidic amino acids (peptidyl‐glutamyl‐peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high‐molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2‐mercaptoethanol dissociates this complex into five low‐molecular weight components. Incubation of the complex at 37°C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl‐glycyl‐glycyl‐leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin‐like activity, exclusively inhibits that activity (Ki= 2.5 × 10−4M), while markedly activating the trypsin‐like activity. The trypsin‐like activity is inhibited by leupeptin (Ki= 1.2 μM) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl‐peptide hydrolyzing activity is activated almost 10‐fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation‐sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high‐molecular weight protein.

Cation-Sensitive Neutral Endopeptidase: Isolation and Specificity of the Bovine Pituitary Enzyme

A highly purified preparation of a cation‐sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS‐containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydro‐phobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH‐RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine‐containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.

PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880‐fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl‐peptide and peptidylprolyl‐amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N‐benzyloxycarbonylglycyl‐L‐prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N‐benzyloxycarbonylglycyl‐L‐proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.

Membrane bound pituitary metalloendopeptidase: Apparent identity to enkephalinase

Abstract A membrane bound zinc-metalloendopeptidase from bovine pituitaries with a specificity toward bonds on the amino side of hydrophobic amino acids, cleaves Met- and Leu-enkephalin at the Gly-Phe bond, releasing Phe-Met and Phe-Leu respectively. The enzyme also hydrolyzes bonds on the amino side of hydrophobic amino acids in oxytocin, bradykinin, neurotensin and several synthetic substrates. A free carboxyl group on a dipeptide C-terminal to the hydrolyzed bond is not a requirement for activity. The enzyme is also present in brain membrane fractions. The regional distribution of this enzyme in brain, its specificity toward natural and synthetic substrates, and its sensitivity to inhibitors, suggest that the enzyme is identical to an activity referred to as “enkephalinase”, which has been described as dipeptidyl carboxypeptidase. The data show that the enzyme is an endopeptidase with a specificity similar to that of a group of microbial proteases, one of which is thermolysin.

Inhibition of Rabbit Brain Prolyl Endopeptidase by N‐Benzyloxycarbonyl‐Prolyl‐Prolinal, a Transition State Aldehyde Inhibitor

Abstract: Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin‐releasing hormone, and luteinizing hormone‐releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl‐prolyl‐proline, with a Ki of 50 μM, prompted the synthesis of benzyloxycar‐bonyl‐prolyl‐prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl‐prolyl‐prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.

A multicatalytical protease complex from pituitary that forms enkephalin and enkephalin containing peptides

Abstract The pituitary contains a high molecular weight (M.W.∼700,000) neutral endopeptidase complex that behaves in solution as a single protein, but on polyacrylamide gel electrophoresis under dissociating conditions shows the presence of five components with molecular weights of 24,000 to 28,000. The complex exhibits trypsinlike, chymotrypsinlike and peptidylglutamyl-peptide bond hydrolysing activities. Experiments indicate that each of these activities is associated with a separate component of the complex. The chymotrypsinlike and trypsinlike activities efficiently generate Leu-enkephalin, Leu-enkephalin-Arg 6 and Leu-enkephalin-Arg 6 -Phe 7 from a single synthetic precursor. Selective inhibition of the chymotrypsinlike activity enhances the trypsinlike activity and alters the relative proportion of opioid peptides formed.

Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.

Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M

Aminopeptidase M (AmM; EC 3.4.11.2) is a membrane-bound peptidase present on renal brush border and vascular plasma membrane. In the present study, AmM, purified from rabbit kidney cortex, produced a single immunoprecipitin line against AmM antisera, hydrolyzed alanyl-, leucyl- and arginyl-beta-naphthylamides at rates of 5.1 +/- 0.5, 3.9 +/- 0.5 and 2.6 +/- 0.3 mumol/min/mg, respectively, exhibited little or no alpha-glutamyl-, aspartyl- or glycyl-prolyl-naphthylamidase activities (less than or equal to 0.14 mumol/min/mg), and was inhibited by o-phenanthroline, amastatin (IC50 = 400 nM) and bestatin (IC50 = 6 microM). The alanyl-naphthylamidase activity of unfractionated rabbit plasma was found to be identical to purified AmM regarding relative rates of hydrolysis of alanyl-, leucyl- and arginyl-naphthylamides (100:79:42), pH optimum, and inhibition profile. In comparative studies with the purified enzyme, immunoreactive AmM accounted for essentially all of the alanyl-2-naphthylamidase activity of rabbit plasma. N-Terminal metabolism of (Met5)enkephalin by purified renal AmM was 3.92 +/- 0.69 mumol/min/mg, followed by somatostatin (1.25 mumol/min/mg), hepta(5-11)substance P (1.14 +/- 0.13 mumol/min/mg), (Asn1)angiotensin II (1.11 +/- 0.06 mumol/min/mg), angiotensin III (0.45 +/- 0.04 mumol/min/mg) and des(Asp1)-angiotensin I (0.36 +/- 0.04 mumol/min/mg). In contrast, substance P, bradykinin, (Sar1,Ala8)angiotensin II and neurokinin analogs containing modified N-termini (e.g. Ac-Arg) were resistant to hydrolysis by AmM. Peptide degradation was optimal at neutral pH and was inhibited by amastatin (IC50 = 200 nM) and bestatin (IC50 = 5 microM). Apparent Km values ranged from 15.7 +/- 0.4 microM for angiotensin III to 102 +/- 2 microM for (Met5)enkephalin. These data support a significant role for vascular and plasma AmM in the metabolism of circulating vasoactive peptides.

Evaluation of dopamine metabolism in rat striatum by a gas chromatographic technique

Dopamine metabolism in rat striatum was evaluated by gas chromatographic quantitation of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The level of DOPAC (5.21 nmoles/g plus or minus 0.40 S.E.M., n equals 12) exceeded that HVA (3.63 nmoles/g plus or minus 0.25 S.E.M., n equals 12). 2 hr following administration of probenecid (200 mg/kg i.p.) the level of striatal HVA was approximately doubled whereas the level of DOPAC was not significantly elevated. Pargyline (75 mg/kg i.p.) poduced a rapid depletion of DOPAC and HVA. the rate of disappearance of DOPAC (t1/2 equals 10 min) exceeded that of HVA (t1/2 equals 18 min). Rates of metabolite formation were computed assuming steady state kinetics. The rate formation of DOPA (20.5 nmoles/g/hr) was much greater than that of HVA (10.1 nmoles/g/hr). We conclude that DOPAC is the major dopamine metabolite in rat striatum and that its measurement may provide the best index of functional neuronal activity in this species.

Delineation of a particulate thyrotropin-releasing hormone-degrading enzyme in rat brain by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase

Abstract: The degradation of thyrotropin‐releasing hormone in rat brain homogenates was studied in the presence of N‐benzyloxycarbonyl‐prolyl‐prolinal and pyroglutamyl diazomethyl ketone, specific and potent active‐site‐directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin‐releasing hormone‐degrading enzyme(s). Reports of a thyrotropin‐releasing hormone‐degrading enzyme with narrow specificity that cleaves the pGlu‐His bond of this tripeptide led us to develop a coupled assay using pGlu‐His‐Pro‐2NA as the substrate to measure this activity. Cleavage of the pGlu‐His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl‐peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o‐phenanthroline. The particulate pyroglutamyl‐peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin‐releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu‐His bond of thyrotropin‐releasing hormone but is distinct from pyroglutamyl peptide hydrolase.