Partial purification and characterization of a putative prohormone-processing enzyme complex from bovine pituitary

A putative prohormone-processing enzyme complex with specificity toward basic residues was partially purified from whole bovine pituitary glands. The complex is basic, binding to S-Sepharose at pH 8.2. The pH optimum of the enzyme is around 8.0. The enzyme is capable of cleaving proenkephalin and is present in at least three forms with relative molecular masses of about 36,000, 58,000, and 90,000 Da. The proteinase complex is inhibited by soybean trypsin inhibitor, limabean trypsin inhibitor, and aprotinin, but not by inhibitors of thiol proteinases or metal chelators. Our results indicate that this proteinase is a trypsin-like serine esterase with properties appropriate to that of a prohormone-processing enzyme.     

Further characterization of a neutral metalloprotease isolated from human articular cartilage

The neutral metalloprotease extracted from 1,200 gm of human articular cartilage was purified 1,400- to 2,400-fold by diethylaminoethyl- and carboxymethyl-Sephadex chromatography. Disc electrophoresis and an isoelectric focusing method resolved the neutral enzyme activity into 4 bands. All bands had a similar amino acid analysis and a similar molecular weight by sodium dodecylsulfate electrophoresis and gel filtration: 24,000–27,000 daltons. The enzyme degraded proteoglycan subunit and proteoglycan aggregate to products with a sedimentation coefficient of 3S, but at low dilutions the enzyme produced 19.3S fragments. It is postulated that this protease may contribute to the development of osteoarthritis from within the cartilage matrix.     

Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase.

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with 51Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of 51Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H2O2; (iii) cytolysis of H2O2-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. We conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase. The participation of enzymes from specific (secondary) granules is excluded because, with the low concentrations of PMA employed, very little release of secondary granule constituents is observed.     

Identification and characterization of brush-border membrane-bound neutral metalloendopeptidases from rat small intestine

Neutral metalloendopeptidase enzymes were identified and partially characterized in the brush-border membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine-4-methoxy-beta-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thirty-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-beta-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brush-border membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function.     

Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses.

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks delta-crystallin/argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, delta-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in epsilon-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.     

Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.     

Diabetic cataract formation: potential role of glycosylation of lens crystallins.

A high glucose concentration in vivo or an increased glucose of glucose 6-phosphate concentration in vitro has been found to lead to the glycosylation of epsilon-amino groups of lysine residues in bovine and rat lens crystallins. In vitro, this glycosylation imparts an increased susceptibility of the crystallins to sulfhydryl oxidation. Disulfide crosslinks result in the formation of high molecular weight aggregates and an opalescence in the crystallin solutions. The addition of reducing agents prevents as well as reverses the formation of high molecular weight aggregates and the opalescence of the crystallins. These phenomena suggest a new interpretation of previous results on cataract formation and a new approach for development of drugs to prevent cataracts.     

The use of affinity matrices in the purification of inhibin from bovine follicular fluid

The protein associated with inhibin-like activity in bovine follicular fluid was purified 80-to 120-fold after successive adsorptions on different affinity matrices, i.e. Matrex gel red A, phenyl sepharose, ω-aminohexyl agarose and concanavalin-A sepharose. Partial characterization of the active protein resulted in the conclusion that inhibin from bovine follicular fluid is a hydrophobic glycoprotein with an apparent molecular weight between 60 000 and 70 000 daltons. An antiserum, raised against an 80-fold purified preparation, prevented the inhibin-like action of bovine follicular fluid on pituitary cells in vitro.     

Isolation, characterisation and tissue localisation of an N-terminal-truncated variant of fibroblast growth factor

An additional form of basic fibroblast growth factor (FGF), possessing full biological activity, has been isolated from bovine pituitary extracts. This polypeptide mitogen exhibits an apparent molecular weight of 17,500 by SDS-PAGE and lacks the first four N-terminal amino acid residues (Pro-Ala-Leu-Pro) of a previously described FGF preparation. Polyclonal antibodies have been raised to this FGF variant and used to examine the distribution of immunoreactive FGF species in rat and bovine tissues by immunoblot procedures. In extracts of rat pituitary, kidney, spleen and liver tissues several higher molecular weight species were detected with immunoreactive 70 kDa and 27 kDa protein bands predominating whilst a 32 kDa immunoreactive protein was also present in the brain. In bovine tissues several immunoreactive proteins with molecular weights greater than 17,500 were also observed in the brain, pituitary, adrenal, ovary and serum with these immunoblot procedures. With bovine pituitary extracts, six immunoblot bands corresponding to immunoreactive proteins of molecular weights 80,000-14,200 were observed, whilst with the brain extracts four immunoblot bands and with serum eight immunoblot bands ranging from 150,000 to 14,200 were detected. With non-immune rabbit serum, no polypeptides/proteins of corresponding molecular weight could be detected with similar immunoblot procedures. The possible relationships of these immunoreactive proteins to FGF precursor forms at various stages of processing are described.     

Inhibition of neutral endopeptidase amplifies the effects of endogenous atrial natriuretic peptide on blood pressure and fluid partition.

Neutral endopeptidase (EC 3.4.24.11) is a wide-spread enzyme that degrades atrial natriuretic peptide (ANP). We studied the effects of a potent neutral endopeptidase inhibitor, SQ 28,603, given intravenously (30 mg/kg over 45 min) to anesthetized, bilaterally nephrectomized Sprague-Dawley rats. Infusion of vehicle alone was accompanied by a modest increase, 3.2 +/- 2.2% (mean +/- SE), in mean arterial blood pressure (MAP) and a slight rise in hematocrit (Hct) of 0.9 +/- 0.7%. After administration of SQ 28,603, MAP fell 3.2 +/- 0.5%, and Hct rose 4.9 +/- 0.5%, both significantly different from the changes with vehicle alone; the lesser increase in plasma protein concentration (2.5 +/- 0.4%) suggested an increase in vascular permeability to both plasma protein and fluid similar to that caused by ANP. When SQ 28,603 was given to rats pretreated with rabbit antirat ANP antiserum, blood pressure rose by 3.8 +/- 0.5%, and Hct increased by 1.0 +/- 0.4%, values very similar to those observed with vehicle alone. Inhibition of neutral endopeptidase therefore amplifies the actions of endogenous ANP on blood pressure and fluid partition.     

Binding of an inhibin-like protein from bull seminal plasma to ovine pituitary membranes

A purified basic protein from bull seminal plasma having inhibin activity was labeled with 125I and tested for binding with ovine pituitary membrane fractions. The bound radioactivity could be eluted under acidic conditions and shown to rebind to fresh pituitary membranes. The properties of the eluted labeled inhibin were similar to the unlabeled fraction. The eluted labeled inhibin exhibited specific binding to the membranes, which was displaceable in a dose-dependent manner by an unlabeled active fraction. Only those fractions in the purification scheme which had inhibin activity also competed for the binding. A bovine follicular fluid fraction with molecular weight greater than 10 000 and inhibin activity also displaced the bound radioactivity from the membranes. Other purified hormones such as ovine FSH, LH or their subunits, prolactin or bovine serum albumin, dialyzed serum or unrelated basic macromolecules such as lysozyme, polylysine, histones, had no influence on the binding of labeled inhibin to ovine pituitary membranes. Synthetic LH-RH also failed to displace the labeled inhibin from the membranes. The binding was sensitive to heat and trypsin treatments. The data are consistent with the direct action of inhibin on the pituitary and demonstrate the existence of binding sites for the active fraction in this target.     

Calcium-activated neutral protease from bovine ventricular muscle: isolation and some of its properties.

A new protease has been isolated and purified from bovine ventricular muscle, by a new method which employs affinity chromatography. This protease required both millimolar concentrations of Ca²⁺ and the SH-group for the activation, and it was active at neutral pH. The molecular weight of this calcium-activated neutral protease was estimated to be 92 000 and 80 000 by gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, respectively. The isoeletric point was determined to be 4.3 by isoelectric focusing. The synthetic substrates including N-benzoyl l-arginine ethylester and acetyltyrosine ethylester were not hydrolysed by this protease. Among the endogenous myofibrillar proteins, the contractile proteins (myosin and actin) were not degraded, while the regulatory proteins, tropomyosin and troponin were degraded only in the presence of Ca²⁺. Of three components of troponin, both troponin-T and troponin-I were susceptible to the proteolytic action of calcium-activated neutral protease, while troponin-C was well preserved, as verified by the electrophoretic pattern in sodium dodecyl-sulfate-polyacrylamide gel. The breakdown of the regulatory proteins was also functional, as demonstrated in natural actomyosin pretreated by calcium-activated neutral protease by measurement of Ca²⁺-sensitivity of superprecipitation. Although the physiological feature of calcium-activated neutral protease is still unknown, this enzyme might act as an aggravating factor in the course of myocardial necrosis, when the intracellular concentration of free Ca²⁺ is considered to be elevated to millimolar levels.     

Preliminary characterization of in vitro synthesized hypothalamic precursor ACTH/beta-endorphin-like material.

We have previously shown that cultured bovine pituitary or hypothalamic cells incorporate 3H-labeled amino acids into high molecular weight glycoproteins containing the antigenic determinants of both corticotropin (ACTH) and beta-endorphin. We now report resolution of this 3H-labeled ACTH/beta-endorphin-like material into two predominant size classes upon SDS polyacrylamide-gel electrophoresis with apparent molecular weights (Mr) of 41 500 +/- 1600 and 36 000 +/- 1100. Isoelectric focusing revealed these components to be basic proteins with apparent isoelectric points greater than 8.5. Overnight trypsinization generated a 3H-labeled fragment comigrating with synthetic beta-lipotropin (61-69) [beta-endorphin (1-9)] upon paper electrophoresis which was immunoprecipitable with antibody directed against the corresponding synthetic fragment. Limited trypsinization of bovine immunoreactive ACTH/beta-endorphin extracted from freshly obtained bovine hypothalamic and anterior pituitary tissue, generated fragments which possessed ACTH bioreactivity. Both bovine pituitary and hypothalamic derived material are similar with respect to these foregoing physiochemical parameters and appear to be larger than the reported forms in mouse pituitary.     

Restorative effect of atrial natriuretic peptide or chronic neutral endopeptidase inhibition on blunted cardiopulmonary vagal reflexes in aged rats

Arterial baroreflex function diminishes with age, but whether cardiopulmonary vagal reflexes are similarly altered with physiological aging has not been fully elucidated. In this study, predominantly cardiac high pressure mechanoreceptor-activated (ramp baroreflex) and cardiopulmonary chemoreceptor-activated (von Bezold-Jarisch reflex) vagal reflexes in conscious, instrumented rats were impaired by 30% to 40% (P<0.05) in 24-month-old (n=12) compared with 6-month-old rats (n=12). To determine whether this is a restorable deficit, the influence of atrial natriuretic peptide (ANP), either by infusion or blockade of its breakdown, was studied. ANP infusion was previously shown to enhance Bezold-Jarisch reflex and ramp baroreflex bradycardia in young adult rats. The present study confirmed that vagal reflex augmentation by ANP (50 pmol/kg per minute) also occurs in old rats (increased by 60+/-18% (Bezold-Jarisch reflex) and 91+/-15% (ramp baroreflex; P<0.05). Direct vagal stimulation in anesthetized animals showed that the target for ANP was not the cardiac vagus itself in old rats (n=7), although in young rats only, we confirmed the published finding that ANP enhances vagal bradycardia (by 58+/-14%, n=7). Neutral endopeptidase 24.11 degrades ANP and several other peptides. The neutral endopeptidase inhibitor candoxatrilat (5 mg/kg per day IV for 7 to 9 days) restored vagal reflex bradycardia in old rats (n=6) to levels similar to those in young neutral endopeptidase inhibitor-treated rats (n=6). Impaired cardiopulmonary vagal reflex control of heart rate is thus a feature of normal aging, and this deficit may be ameliorated by either ANP infusion or chronic neutral endopeptidase inhibition.     

Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase

Metabolism of natriuretic peptides is regulated by two degradative pathways: uptake by the clearance receptor (natriuretic peptide receptor C--NPR-C) and hydrolysis by neutral endopeptidase (NEP). Affinity studies favour a dominant role of NPR-C in hormone degradation in several species but do not account for the efficacy of NEP inhibitors in vivo, nor the uniquely prolonged half life (t((1/2))) of human brain natriuretic peptide (hBNP). Postulating that (1) delayed metabolism of hBNP reflects resistance to NEP and (2) interactions between NPR-C and NEP increase enzyme activity, we have used purified ovine and human NEP, plus ovine lung plasma membranes to study the relative importance of receptor and enzyme pathways. We have also related the findings to hormone metabolism in vivo. Binding affinities of atrial natriuretic peptide (ANP), hBNP and ovine BNP (oBNP) to oNPR-C were similar (K(d)=8-16 pM). In contrast, unlike ANP and oBNP, hBNP was not significantly degraded by purified oNEP or plasma membranes. Despite similar (and high) affinity of oNPR-C for oBNP and hBNP, the t((1/2)) of hBNP (12.7 min) was more than fourfold that of oBNP (2.6 min). Although we found no evidence for receptor-enzyme interaction, our results show that the delayed metabolism of hBNP reflects resistance to NEP. These findings have important implications for future treatment strategies in human disease.     

Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones.

A porcine kidney microsomal metalloendopeptidase has been enriched 3900-fold. Gel filtration on a calibrated Toyo-Soda G-3000 SW column indicated an appropriate molecular weight for the endopeptidase of 88,000 +/- 2000. The purified enzyme is inhibited by a number of synthetic inhibitors of thermolysin. The endopeptidase hydrolyzes the succinyl (Suc)-containing fluorogenic peptide substrate Suc-Ala-Ala-Phe-(7-amino-4-methylcoumarin) at the Ala-Phe position with a Km of 2.9 X 10(-4) M. The endopeptidase also hydrolyzes a variety of peptides including corticotropin, substance P, angiotensin I and II, neurotensin, somatostatin, bradykinin, and the renin tetradecapeptide substrate. The endopeptidase hydrolyzes both [Leu]- and [Met]enkephalin at the Gly-Phe bond.     

Processing of the precursor to corticotropin and beta-lipotropin in humans.

The biosynthesis of human corticotropin (ACTH) was studied in organ culture of pituitary adenomas and by translating mRNA from an ectopic ACTH-producing tumor in a cell-free system. Peptides similar to human ACTH, beta-lipotropin, and the amino-terminal glycopeptide are cleaved from a common precursor with an apparent molecular weight of 35,000 on sodium dodecyl sulfate/polyacrylamide gels. The precursors synthesized in pituitary and ectopic ACTH-producing tissues are indistinguishable. The cleavage sites of the peptide chain appear to be similar to those previously deduced for murine and bovine ACTH. Immunoprecipitation studies suggest that the primary structure of the precursor peptide is similar in all three species. However, glycosylation is different in the human and murine precursors: the precursor to human ACTH appears to be glycosylated only in the amino-terminal fragment, not in the ACTH or beta-lipotropin sequences. Studies with an autopsied normal human pituitary suggest that neither normal nor adenomatous pituitary tissue glycosylates the ACTH sequence.     

Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei.

Two type I DNA topoisomerases have been purified to homogeneity from the nuclei of HeLa cells. One topoisomerase has a peptide molecular weight of 100,000 and the other, a molecular weight of 67,000. Several lines of evidence indicate that these two topoisomerases are closely related, (a) Both exhibit similar enzymatic activities on DNA. (b) The chromatographic properties of the two topoisomerases during purification are similar. (c) Mild proteolysis of the purified molecular weight 100,000 topoisomerase in vitro generates a group of protein bands of molecular weight approximately 67,000, and these bands retain topoisomerase activity. (d) The peptides formed by partial proteolysis of the 67,000 topoisomerase in the presence of NaDodSO4 form a subset of those produced from the 100,000 enzyme. The 100,000 topoisomerase is the major type I enzyme in the cell. The 67,000 topoisomerase, which may be identical to the previously identified "nicking-closing" enzyme [Champoux, J. J. & Dulbecco, R. (1972) Proc. Natl. Acad. Sci. USA 69, 143-146], is probably formed by proteolysis of the 100,000 enzyme.     

Degradation of porcine relaxin by glutathione-insulin transhydrogenase and a neutral peptidase

The susceptibility of porcine relaxin and 125I-polytyrosyl-porcine relaxin to degradation by 3 purified enzymes involved in the degradation of insulin and proinsulin was examined. Rat liver glutathione-insulin transhydrogenase (GIT), which cleaves disulfide bonds in insulin, catalyzed a time- and concentration-dependent increase in trichloroacetic acid (TCA)-soluble radioactivity of relaxin. The Sephadex G-50 profile of the reaction products revealed conversion to the A- and B-chains. Relaxin competitively inhibited the degradation of insulin by GIT; however, kinetic analysis revealed insulin to be preferred over relaxin as a substrate. Rat liver cytosol neutral thiol peptidase (NTP) catalyzed a time- and concentration-dependent increase in the TCA solubility of relaxin and a shift in the Sephadex G-50 radioactivity profile to low molecular weight products. Kinetic analysis revealed that insulin and B-chain are preferred over relaxin as substrates for NTP. A third enzyme, rat kidney neutral metalloendopeptidase, which degrades proinsulin and insulin C-peptide but not insulin, also did not degrade porcine relaxin.     

A Bovine Pancreatic Enzyme Catalyzing the Conversion of Proinsulin to Insulin

An enzyme that catalyzes the conversion of bovine proinsulin to insulin has been purified from a bovine pancreatic extract. The product of conversion was identified as insulin by aminoacid analysis and determination of carboxyl terminal aminoacid residues. The purified enzyme preparation showed one major protein band on polyacrylamide gel disc electrophoresis; it had a molecular weight of about 70,000 and an isoelectric point (pI) at a pH of 4.82.