Preparation and characterization of an immuno-electron microscope tracer consisting of a heme-octapeptide coupled to fab.

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent K(m) of 0.2 M, an apparent v(max) of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of approximately 2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 A, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent K(m) of 0.4 M, an apparent v(max) of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

Preparation of functional smooth muscle cells from the rabbit aorta.

A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.

Reversal of experimental autoimmune encephalomyelitis with a hydroxamate inhibitor of matrix metalloproteases.

Gelatinases, belonging to the matrix metalloproteases, contribute to tissue destruction in inflammatory demyelinating disorders of the central nervous system such as multiple sclerosis. We used experimental autoimmune encephalomyelitis (EAE) as an animal model to evaluate the effect of a hydroxamate matrix metalloprotease inhibitor (GM 6001) on inflammatory demyelination. A single dose of the inhibitor, given intraperitoneally, provided sufficient levels in the cerebrospinal fluid of animals with EAE to induce at least a partial inhibition of the gelatinase activity in the cerebrospinal fluid. When administered daily either from the time of disease induction or from the onset of clinical signs, GM 6001 suppressed the development or reversed clinical EAE in a dose-dependent way, respectively. Animals returned to the same clinical course as the nontreated group after cessation of treatment. Animals treated from the onset of clinical signs had normal permeability of the blood-brain barrier, compared with the enhanced permeability in nontreated animals. These results indicate that matrix metalloprotease inhibition can reverse ongoing EAE. This effect appears to be mediated mainly through restoration of the damaged blood-brain barrier in the inflammatory phase of the disease, since, the degree of demyelination and inflammation did not differ between the treatment groups.

Inhibition of angiogenesis by the matrix metalloprotease inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L-tryptophan methylamide.

The inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L- tryptophan methylamide specifically blocks several matrix metalloproteases, enzymes which are thought to be involved in angiogenesis. An extract of Walker 256 carcinoma in Hydron pellets implanted in the corneas of Sprague-Dawley rats was used to stimulate angiogenesis from the vessels of the limbus. Angiogenesis was graded visually as the distance penetrated into the cornea and the number of vessels generated. The vessel area was also measured by image analysis using Image 1 software. Continuous i.v. administration of N-[2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]- L-tryptophan methylamide at 32 mg/kg/day (n = 17) via syringe pump reduced vessel number [25.06 +/- 5.9 (SEM) compared to 65.33 +/- 9.0] and vessel area (26.14 +/- 3.2 mm2 compared with 40.96 +/- 4.6 mm2), but not distance penetrated, compared to vehicle-treated control eyes after 6 days. These results confirm the suspected role for matrix metalloproteases in angiogenesis and suggest that inhibitors of these enzymes may be angiostatic agents.

Matrix metalloproteinase expression in human retinal microvascular cells.

The degree of hyperglycemia correlates with the development of diabetic retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.

Photochemical inactivation of the angiotensin receptor of rabbit aorta by N alpha-(2-nitro-5-azidobenzoyl)-[1-aspartic acid,5-isoleucine]angiotensin II.

The photoaffinity label N alpha-(2-nitro-5-azidobenzoyl)-[Asp1,Ile5]angiotensin II (nitroazidobenzoyl-angiotensin II) has a pD2 of 7.22 in the rabbit aortic strip assay compared to 8.44 for [Asp1,Ile5]angiotensin II and can be completely inhibited by [Sar1, Ala8]angiotensin II. Photolysis of a single dose of nitroazidobenzoyl-angiotensin II (0.1 microM) with aortic strips for 3 min with light of wavelengths of greater than 320 nm, followed by washing, resulted in loss of approximately 25% of the strip's response to a subsequent dose of nitroazidobenzoyl-angiotensin II. Photolysis of three consecutive doses of label resulted in loss of 62% of the strips response to a subsequent dose. Repetitive exposure to label in the dark and repetitive photolyses without label resulted in loss of no more than 20% of the strips ability to respond to angiotensin II. Photolysis with three consecutive doses of 0.1 microM nitroazidobenzoyl-angiotensin II in the presence of 0.5 microM of the competitive inhibitor [Sar1,Ala8]angiotensin II decreased the observed inhibition from 62 to 22%. The long-lasting inhibition observed after photolysis with 0.1 microM nitroazidobenzoyl-angiotensin II therefore occurred via photolysis of some part of the nitroazidobenzoyl chromophore. This inhibition was mediated via the receptor that binds [Sar1,Ala8]angiotensin II and, therefore, angiotensin II.

Phosphoramidate peptide inhibitors of human skin fibroblast collagenase.

An extensive series of N-(monoethylphosphoryl)peptides was synthesized and their inhibition of purified human skin fibroblast collagenase examined. At the cleavage site S1 all reported compounds have the (EtO)(OK)P(O) group and the peptide side chain extended toward the C-terminal end (up to P5') of the substrate sequence. These phosphoramidates with a tetrahedrally hybridized phosphorus atom are thought to be transition state analogue inhibitors. They exhibited fair inhibitory potency against this vertebrate collagenase having Ki values in the micromolar range. The most potent of these, (EtO)(OK)P(O)-Ile-TrpNHCH3 (68), inhibits with a Ki value of 1.5 microM and is nearly 100 times stronger than (EtO)(OK)P(O)-Ile-Ala-GlyOK (51) (Ki of 140 microM), which has the sequence matching that of the alpha 1 (I) chain of collagen in P1', P2', P3' after the cleavage site. Several compounds were prepared in an attempt to identify the nature of the S2', S3', and S4' binding sites. Alanine at the P2' position was replaced by leucine, phenylalanine, tryptophan, or tyrosine derivatives, resulting in Ki values in a significantly lower range, 1.0-40 microM, compared to 51. No upper size limitation or specificity has been found at this position, yet similar replacements at the P3' position, which is occupied naturally by a glycine residue, gave weaker inhibitors: (EtO)(OK)P(O)-Ile-Tyr(OBzl)-PheOK (57) had a Ki of 120 microM. Hexapeptide derivatives had weaker activities in the 270 microM-2 mM range. All inhibitors were evaluated by using the synthetic thio peptolide spectrophotometric assay.

N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline, N alpha-[bis (4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline, and N alpha-[ (2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline: releasers of potent inhibitors of angiotensin converting enzyme at physiological pH and temperature.

The rate of loss of phenol or 4-nitrophenol from N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline (2), N alpha-[bis(4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline (5), and N alpha-[(2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline (12) was determined spectrophotometrically at pH 7.5 and 37 degrees C in both Tris and phosphate buffers. These moderately potent inhibitors of angiotensin converting enzyme (Ki greater than 0.8 microM) all hydrolyze, losing 1 mol of phenol to yield highly potent inhibitors (Ki = 0.5-18 nM). The half-times for loss of 1 mol of phenol in Tris buffer are 22 days (2), 3.4 h (5), and 21 days (12). The half-times in phosphate buffer were not significantly different. The mono(4-nitrophenoxy) ester 6 (Ki = 18 nM) loses its 1 mol of nitrophenol with a half-time of 35 h to yield N alpha-phosphoryl-L-alanyl-L-proline 16 (Ki = 1.4 nM), which hydrolyzes at the P-N bond with a half-time of 2.2 h. Hydrolysis of the P-N bond in 2 and 12 was not observed during the time course of the kinetic experiments. The two phosphoramidate diesters 2 and 5 and the phosphonamidate monoester 12 thus release powerful inhibitors of angiotensin converting enzyme with a known time course at physiological pH and temperature in vitro. A time-dependent increase in inhibitory potency against converting enzyme that paralleled the kinetics of phenyl ester hydrolysis was confirmed in vitro.

Matrix metalloproteinase inhibitors: a structure-activity study.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

An inhibitor of the matrix metalloproteinase synthesized by rabbit corneal epithelium.

Normal and abnormal processes of cellular invasion often are initiated by degradation of basement membranes. The process of corneal ulceration might operate via similar mechanisms; degradation of the corneal stroma is not seen until after the basement membrane underlying the corneal epithelium in the preulcerative lesion is lost. Recent data implicate a member of the matrix metalloproteinase (MMP) family of enzymes, 92 kD gelatinase/type IV collagenase (MMP-9) in both cellular invasion processes and degradation of epithelial basement membrane before corneal ulceration. This suggests that use of nontoxic substances that block activity of MMP-9 might be useful in preventing or inhibiting pathologic invasion processes in vivo. An agent that fits these criteria is N-[D,L-2-isobutyl-3(N'-hydroxycarbonylamido)-propanoyl]-O- methyl-L-tyrosine methylamide, which previously has been characterized as an inhibitor of tumor cell collagenases. In this study, the authors show that the inhibitor can efficiently block activity of MMP-9 purified from cultures of rabbit corneal epithelial cells. Results suggest that the recently reported efficacy of a closely related inhibitor in blocking progression of alkali burns to ulceration might be attributable to its action against MMP-9.

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Administration of a matrix metalloproteinase inhibitor after hemorrhage improves cardiovascular and hepatocellular function.

Although matrix metalloproteinase inhibitors prevent the increase in soluble tumor necrosis factor-alpha during endotoxemia, it remains unknown whether a novel matrix metalloproteinase inhibitor, GM6001, improves cardiovascular and hepatocellular function after trauma and hemorrhage. To determine this, rats underwent laparotomy (i.e., trauma-induced), and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximal shed volume was returned in the form of Ringer's lactate. The animals were then resuscitated with 3 times the volume of maximal bleedout with Ringer's lactate over 45 min, followed by 2 times Ringer's lactate over 60 min. GM6001, at a dose of 100 mg/kg or an equal volume of normal saline, was administered subcutaneously 15 min before the completion of resuscitation. At 2 and 4 h after resuscitation, cardiac output was measured by indocyanine green (ICG) dilution. Hepatocellular function (i.e., maximum velocity and the efficiency of ICG clearance) was determined by in vivo ICG clearance. Microvascular blood flow in various organs was assessed by laser Doppler flowmetry. The results indicate that cardiac output, hepatocellular function, and tissue microvascular blood flow decreased significantly at 2 and 4 h after resuscitation. GM6001 treatment, however, significantly improved the depressed cardiovascular and hepatocellular function. Since GM6001 improves cardiovascular and hepatocellular function, this agent may be a useful adjunct to fluid resuscitation after trauma and hemorrhagic shock.

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality.

Excessive tumor necrosis factor alpha (TNF alpha) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNF alpha production rely on monoclonal antibodies or immunoadhesins that bind TNF alpha and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNF alpha processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 microgram/mL endotoxin, GM-6001 at concentrations > 5 micrograms/mL blocked release of TNF alpha, but did not affect the release of either IL-1 beta or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNF alpha. To confirm the role of secreted TNF alpha in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 micrograms/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNF alpha response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1 beta and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNF alpha processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNF alpha both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

Localization of secretory IgA, secretory component, and alpha chain in the mammary gland of lactating rabbits by immunoelectron microscopy.

The different steps in the processing of secretory IgA (sIgA) of the mammary gland of lactating rabbits have been investigated with immunoelectron microscopy. The method employed consists of an indirect localization sequence, in which the immunologic reagents are allowed to diffuse into the fixed tissue prior to embedding and thin sectioning. In the first step, goat F(ab')2 fragments directed against sIgA, secretory component (SC), or alpha chain were used. To visualize the location of these first-step antibody fragments, a second Fab fragment directed against goat F(ab')2 antibody was prepared and coupled to a small heme octapeptide that possessed peroxidatic activity, the reaction product of which is visible by electron microscopy. Both the epithelial cell and the local plasmacytes of the mammary gland contain alpha chain, whereas SC is exclusively located in the epithelial cells. In the plasmacytes, both the cisternae of the rough endoplasmic reticulum and the saccules and vesicles of the Golgi complex contain alpha chain. In the epithelial cells, the elements of the Golgi complex and large apical vacuoles situated in the apical region of the cell. Based on these results, a model for processing of sIgA is proposed and discussed.

Matrix metalloproteinase inhibitors reduce phorbol ester-induced cutaneous inflammation and hyperplasia.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases which play key roles in extracellular matrix remodeling, connective tissue damage, inflammation and cell proliferation in a variety of tissues. Since MMP inhibitors have been recently shown to decrease proliferation of vascular smooth-muscle cells, and to prevent neutrophil infiltration in response to alkali burns, we sought to determine whether MMPs play a role in the pathogenesis of inflammatory or hyperproliferative skin disorders. The effects of a specific MMP inhibitor and its analogues on phorbol dibutyrate (PdiBu)-induced inflammation and epidermal hyperplasia in murine skin were assessed. Topical GM 6001, a hydroxamic acid analog with potent inhibitory activity against several MMPs, markedly inhibited PdiBu-induced increases in both ear thickness and ear punch-biopsy weight in a dose-dependent manner 30 h after topical application of PdiBu. Maximal inhibition (75%) was obtained at a dose of 100 micrograms/cm2 (P < 0.01). Moreover, histologic analysis revealed that GM 6001 decreased both the inflammatory cellular infiltrates and epidermal hyperplasia induced by PdiBu. Whereas similar results were found for GM 1489, an analog of GM 6001, acetohydroxamic acid, containing the critical metal ligand group but without the amino acid side chains necessary for binding to the MMPs, did not alter the response to PdiBu inflammation/hyperplasia. These results show that the MMP inhibitors, GM 6001 and GM 1489, are effective in reducing both the inflammatory and hyperproliferative responses that occur following topical phorbol ester application, suggesting a potential role for MMPs in cutaneous inflammatory dermatoses. Moreover, the delivery of this class of inhibitors across intact stratum corneum implies that MMP inhibition could provide an approach to the topical treatment of inflammatory dermatoses.

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates.

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues.

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme.

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)