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A G Scicli

Publications and source records attributed to A G Scicli.

At least 55 records · Page 3Linked to original sources
PubMed1991

Cosegregation of blood pressure with a kallikrein gene family polymorphism.

It has recently been proposed that sequence variation in the gene coding for tissue kallikrein might be involved in the pathogenesis of hypertension. However, molecular evidence of an association between a sequence alteration in the kallikrein gene family and the transmission of increased blood pressure has never been reported. In 32 recombinant inbred (RI) strains derived from the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway rat (BN), we investigated whether a restriction fragment length polymorphism (RFLP) marking the kallikrein gene family cosegregated with blood pressure. In the RI strains that inherited the kallikrein RFLP from the SHR progenitor strain, the median systolic, diastolic, and mean arterial pressures were significantly greater than in the RI strains that inherited the kallikrein RFLP from the BN progenitor strain. These findings suggest that in the rat, sequence variation in the kallikrein gene family, or in closely linked genes, may have the capacity to affect blood pressure.

Animals↗
PubMed1991

Zinc metallopeptidase inhibitors. A novel antihypertensive treatment.

Inhibitors of two zinc metallopeptidases, angiotensin I converting enzyme (ACE) and neutral metalloendopeptidase-24.11 (EP-24.11), are antihypertensive agents. In this issue of Hypertension, Genden and Molineaux report that yet another peptidase inhibitor, metalloendopeptidase-24.15, EC 3.4.24.15 (EP-24.15), lowers blood pressure in normotensive rats. In this editorial we discuss the possible role of kinins as common mediators of part of the vasodepressor action of these peptidase inhibitors. Genden and Molineaux report that the marked fall in blood pressure caused by the EP-24.15 inhibitor is almost abolished by a kinin receptor antagonist, supporting the hypothesis that kinins play a role in the regulation of normal blood pressure. We have confirmed that the EP-24.15 inhibitor used by these investigators lowers blood pressure. Up to now, EP-24.15 has not been implicated in in vivo metabolism of kinins. Although a number of kininases have been identified, our own previous work indicated that the metabolic pathway responsible for clearing kinins from the circulation involves the action of kininase II (angiotensin I converting enzyme) and renal peptidases. Nevertheless, the main metabolic pathway involved some other unidentified enzyme, since in these experiments disappearance of kinins from the circulation was only marginally reduced by a "cocktail" of inhibitors of ACE, EP-24.11, and carboxypeptidase N. It could be that EP-24.15 is involved in kinin metabolism. However, a number of questions need to be answered with regard to the mechanism by which the EP-24.15 inhibitor lowers blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Local hormonal factors (intracrine, autocrine, and paracrine) in hypertension.

Vasoactive hormones acting as endocrine, neuroendocrine, or local hormonal systems (intracrine, autocrine, and paracrine) are an important component of the many factors that regulate blood pressure. Hypertension may be the result of an alteration in the balance between vasodepressor and vasopressor hormonal systems. Changes in this balance could be due to genetic factors such as mutations in one of the genes of the vasoactive system or environmental factors that alter the synthesis and release of one or more vasoactive hormones. Endocrine and neuroendocrine vasopressor hormonal systems, such as the renin-angiotensin system and catecholamines, play a well-established and important role in the regulation of blood pressure and the pathogenesis of some secondary forms of hypertension. The blockade of such systems has already resulted in effective antihypertensive treatment. The role of local hormonal systems is less well established; however, recent evidence suggests they also play an important role in the regulation of blood pressure and the pathogenesis of hypertension. Some vasopressor hormonal systems, such as the renin-angiotensin system, can act as both endocrine or local hormonal systems. Work using transgenic rats harboring the mouse Ren-2 gene has conclusively demonstrated that the renin-angiotensin system, acting as a local hormonal system, has the capability to cause severe hypertension. Whether this model of experimental hypertension mimics any type of human hypertension is not known. Vasodepressor hormones such as kinins, prostaglandins, and endothelium-derived relaxing factor (EDRF) act mainly as local hormonal systems, with the notable exception of atrial natriuretic factor, which may act as both an endocrine and a local hormone. The tissue kallikrein-kinin system, acting either directly or via paracrine eicosanoids or EDRF, participates in local regulation of the circulation, renal function, and the acute antihypertensive effect of angiotensin converting enzyme inhibitors. A restriction fragment length polymorphism (RFLP) that distinguishes the kallikrein gene family of a strain of spontaneously hypertensive rats (SHR) from normotensive Brown Norway rats has been identified. In a set of 32 recombinant inbred strains derived from these SHR and Brown Norway strains, the RFLP marking the kallikrein gene family of SHR cosegregated with an increase in blood pressure. Also, in a study of Utah families it was found that a dominant-allele kallikrein gene expressed as high urinary kallikrein excretion was associated with a decreased risk of essential hypertension. In conclusion, vasopressor and vasodepressor hormones, acting not only as endocrine but also as local hormones, play an important role in the regulation of blood pressure and the pathogenesis of hypertension.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin II↗
PubMed1990

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase).

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.

Amino Acid Sequence↗
PubMed1990

Renal effects of Fab fragments of kinin antibodies on deoxycorticosterone acetate-salt-treated rats.

The role of the renal kallikrein-kinin system in the regulation of renal function is not completely understood. Intrarenal kinins can influence renal function by acting as paracrine hormones at basolateral, luminal, or both sites in the distal nephron. To examine the role of intrarenal kinins in deoxycorticosterone acetate-salt-treated rats, which have high renal kallikrein, Fab fragments of antibradykinin antibody or DArg[Hyp3Thi5,8DPhe7]bradykinin, a kinin antagonist, were used to block kinins. At the dose used, the antibody (25 mg) and kinin antagonist (10 micrograms/min/rat) inhibited the hypotensive effect of intra-arterially injected bradykinin (100 ng) by 70% and 52%, respectively. The antibody appeared in the urine within 30 minutes after administration. Urinary volume was lowered from 9.4 +/- 0.2 to 6.7 +/- 0.4 microliters/min/g kidney wt (p less than 0.001, paired t test) by the antibody and from 8.5 +/- 0.3 to 6.8 +/- 0.4 microliters/min/g kidney wt (p less than 0.004, paired t test) by the kinin antagonist. The antibody lowered urine sodium excretion from 1.11 +/- 0.04 to 0.88 +/- 0.06 mueq/min/g kidney wt (p less than 0.001, paired t test), whereas the kinin antagonist had no significant effect. Neither altered blood pressure, renal blood flow, or glomerular filtration rate. These data suggest that in deoxycorticosterone acetate-salt-treated rats, excretion of water and sodium is regulated in part by kinins. The antidiuretic effect of the antibody and kinin antagonist might be due to blockade of kinins in the vascular-interstitial space of the kidney, since the kinin antagonist is likely hydrolyzed in the proximal tubule and does not reach the lumen of the distal nephron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A kallikrein-like enzyme in blood vessels of one-kidney, one clip hypertensive rats.

Active and inactive kallikrein or a kallikrein-like enzyme are found in the aorta, vena cava, and tail artery and veins of the rat. We studied the concentration of vascular kininogenase in rats with one-kidney, one clip renovascular hypertension and in unilaterally nephrectomized normotensive rats. Six weeks after surgery, active and total vascular kininogenase activity (active plus trypsin-activated) was measured. Blood pressure was 212 +/- 4 mm Hg in the hypertensive rats (n = 33) and 120 +/- 1 mm Hg in the normotensive rats (n = 32) (p less than 0.001). Active kininogenase was lower in the hypertensive rats; although the difference was not significant in the thoracic aorta (56 +/- 8 versus 77 +/- 15), it was highly significant in the abdominal aorta (63 +/- 13 versus 167 +/- 17, p less than 0.001) and tail artery (48 +/- 8 versus 197 +/- 31, p less than 0.003). Total vascular kininogenase activity (active plus trypsin-activated) was lower in the hypertensive rats in all arteries examined: thoracic aorta (183 +/- 16 versus 380 +/- 38, p less than 0.003), abdominal aorta (565 +/- 61 versus 1,093 +/- 74, p less than 0.001), and tail artery (532 +/- 112 versus 1,243 +/- 135, p less than 0.003). Active kininogenase in the vena cava was higher in the hypertensive rats (213 +/- 56 versus 131 +/- 31); however, this difference was not statistically significant, whereas in the tail veins it was highly significant (1,803 +/- 221 versus 771 +/- 79, p less than 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Kallikrein messenger RNA in rat arteries and veins.

Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effects on blood pressure of intracerebroventricular administration of a kinin antagonist.

The components of the kallikrein kinin system are present in brain, and blood pressure is increased when kinins are injected into the brain cerebral ventricles. Therefore, brain kinins may play a role in the regulation of blood pressure. Kinin analogs which are effective as blockers of the vasodilatory and musculotropic activity of kinins have recently become available. Our objective in this study was to examine whether one of these kinin antagonists (K-ant) inhibits the pressor activity of intracerebroventricular (ICV) bradykinin, and whether ICV injections of this antagonist would alter basal mean blood pressure (BP). The analog used was DArg0-Hyp3-Thi5,8-DPhe7-bradykinin. All injections (10 microliters) were made into a lateral cerebral ventricle of restrained, conscious rats. The hypertensive response to 0.5 microgram bradykinin was partially blocked by simultaneous injections of either 5 or 10 micrograms of the K-ant: From (delta MBP = 36 +/- 7 to 17 +/- 5, p less than 0.05, and from 34 +/- 6 to 8 +/- 1 mmHg, p less than 0.01, respectively. Lower concentrations of K-ant were ineffective; the K-ant at doses up to 10 micrograms had no effect on BP. Doses of 25 and 50 micrograms increased BP (delta MBP = 32.5 +/- 5 and 59.0 +/- 5.5 mmHg, respectively, p less than 0.001). Both the 25 and the 50 micrograms doses of the K-ant induced barrel rotation in all rats. These data suggest that kinins present in circumventricular structures do not play a role in normal blood pressure regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Glandular kallikrein-like enzyme in adrenal glands.

A kallikrein-like kininogenase was identified in the rat adrenal gland. Most of the enzyme was present in an inactive form, since pre-incubation with trypsin markedly increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23.0 pg bradykinin/mg protein/min. Adrenal kininogenase was inhibited 90% by phenyl methyl sulfonyl fluoride, 92% by D-Phe-Phe-Arg-chloromethylketone, 91% by aprotinin, and only 15% by soybean trypsin inhibitor. Pre-incubation with antibodies against rat urinary kallikrein resulted in 85% inhibition. The apparent molecular weight of adrenal kininogenase on gel filtration chromatography was 33 Kd. The enzyme was strongly adsorbed to immobilized rat urinary kallikrein antibodies and required drastic conditions for elution. In canine adrenal glands, we found that there was no difference in the cortical and medullary distribution of active and inactive SBTI resistant kininogenase activity. We conclude that an enzyme which closely resembles glandular kallikrein is present in adrenal glands.

Adrenal Cortex↗
PubMed1989

Role of kinin in regulation of rat submandibular gland blood flow.

In tissues rich in kallikrein, vasodilator kinins, acting as paracrine hormones, may play a role in the local regulation of blood flow. We studied the role of kinins in the regulation of blood flow in the rat submandibular gland using a kinin analogue with antagonistic properties, [DArg0]Hyp3-Thi5-8[DPhe7]bradykinin. When infused into the carotid artery (20 micrograms/min/rat), this antagonist blocked the effect of bradykinin (25-250 ng/kg, intracarotid injection) on glandular blood flow. In nephrectomized rats, the antagonist also blocked the increase in glandular blood flow caused by enalaprilat, a kininase II converting enzyme inhibitor. At a dose of 20 micrograms/min/rat, the antagonist produced no detectable change in basal glandular blood flow; however, at a higher dose (100 micrograms/min/rat), it caused a significant decrease (p less than 0.001). In eight of 10 rats, blood flow decreased by 75% or more; this effect was not blocked by the alpha-adrenergic receptor antagonist phentolamine. After antagonist infusion was stopped, blood flow returned toward normal. Sympathetic nerve stimulation of the gland induced vasoconstriction followed by poststimulatory vasodilatation. In rats displaying severe vasoconstriction after the antagonist, postsympathetic vasodilatation was abolished even when stimulation was performed after the antagonist infusion had been stopped and blood flow returned toward normal. Although a direct vasoconstrictor effect of the kinin antagonist cannot be completely ruled out, these data suggest that, in the rat submandibular gland, kinins may play a role in regulation of basal blood flow and vasodilatation after converting enzyme inhibitor or sympathetic stimulation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1989

Role of angiotensin converting enzyme and other peptidases in in vivo metabolism of kinins.

Arterial plasma kinins and mean arterial pressure were measured in intact and bilaterally nephrectomized rats infused with vehicle or bradykinin to study the role of 1) angiotensin converting enzyme (ACE) and other peptidases and 2) the kidney (a kininase-rich organ) in the metabolism of kinins in vivo. Before the infusion, rats were pretreated with vehicle, enalaprilat (an ACE inhibitor), or a cocktail of kininase inhibitors containing 1) enalaprilat, 2) DL-2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid (MGTA), a carboxypeptidase N inhibitor, 3) phosphoramidon, a neutral endopeptidase 24.11 inhibitor, and 4) bestatin, an aminopeptidase B inhibitor. In the rats with vehicle (n = 8), the cocktail did not significantly increase endogenous kinins (from 31 +/- 6 to 41 +/- 9 pg/ml, p = 0.94). In the rats infused with bradykinin (peptidase substrate), plasma kinins increased threefold in the group pretreated with the vehicle, 21-fold in the enalaprilat group, and 22-fold in the cocktail group. These increases were doubled by nephrectomy but were not affected by ureteral ligation. In the groups pretreated with the cocktail or enalaprilat, the hypotensive effect of bradykinin was correlated with plasma kinin concentration (r = 0.75, p less than 0.001). After bradykinin infusion was stopped, plasma kinins decreased by half in 10-12 seconds in the rats pretreated with vehicle, enalaprilat, or cocktail. We concluded that ACE and the kidney are important to the metabolism of circulating kinins while carboxypeptidase N, neutral endopeptidase 24.11 and aminopeptidase B are not. We also concluded that other tissue peptidases, not affected by either the above inhibitors or nephrectomy, play an important role in kinin metabolism.

Animals↗
PubMed1989

Role of endogenous brain kinins in the cardiovascular response to intracerebroventricular melittin.

Intracerebroventricular infusion of the peptide melittin increases immunoreactive kinins in the cerebrospinal fluid of anesthetized dogs, probably secondary to activation of brain or cerebrospinal fluid kininogenases. Intracerebroventricular melittin also increases blood pressure and heart rate, possibly mediated by brain kinins, since intracerebroventricular bradykinin also increases blood pressure and heart rate. We tested whether the effects of centrally administered melittin on blood pressure and heart rate could be blocked by simultaneous infusion of a kinin receptor antagonist, [DArg0]Hyp3-Thi5,8[DPhe7]bradykinin, in normotensive awake rats. In the controls, intracerebroventricular infusion of kinin receptor antagonist given for 1 hour at a rate of 10 micrograms/hr blocked bradykinin-induced increases in blood pressure and heart rate by 80%. Basal blood pressure and heart rate were not affected by the kinin receptor antagonist alone. After a 30-minute infusion of melittin (8 micrograms/30 min), cerebrospinal fluid kininogenase activity (n = 17) rose from 0.13 +/- 0.05 to 0.43 +/- 0.1 ng/ml/min (p less than 0.02). Although cerebrospinal fluid kinins increased from below sensitivity (0.02 ng/ml, n = 12) to 0.19 +/- 0.1 ng/ml (n = 17), this change was due to drastic increases in three rats, whereas in 12 of them kinins were below sensitivity. Incubation of bradykinin (10 ng) with 0.1 ml rat cerebrospinal fluid for 5 minutes destroyed 70% of kinins, suggesting that rapid destruction may have made detection of increased CSF kinins difficult.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Contributions of various rat plasma peptidases to kinin hydrolysis.

The relative contribution of plasma carboxypeptidase N (kininase I), angiotensin-converting enzyme (ACE) (kininase II), neutral endopeptidase 24.11 (enkephalinase A) and postproline cleaving enzyme to total kininase activity in rat plasma was determined by measuring bradykinin hydrolysis with and without various concentrations of inhibitors of these enzymes. We used DL-2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid to inhibit kininase I, enalaprilat for ACE, phosphoramidon for neutral endopeptidase 24.11 and N-benzyloxycarbonyl-Pro-prolinal for postproline cleaving enzyme. Bradykinin was added to rat plasma and incubated at 37 degrees C. Kininase activity was evaluated based on the decrease in bradykinin during incubation. Bradykinin was measured by radioimmunoassay, using an antibody that recognizes its carboxyl group. Of the total plasma kininase activity, carboxypeptidase N was responsible for 11.0 +/- 2.5% (N = 5; P less than .05) and ACE for 46.8 +/- 1.5% (N = 5; P less than .001), whereas the contribution of neutral endopeptidase 24.11 and postproline cleaving enzyme turned out to be negligible. Of the kininase activity in rat plasma, 42% could not be explained by any of these four enzymes. We concluded that ACE is responsible for most of the kininase activity in rat plasma; carboxypeptidase N contributes to a slight degree. The fact that 42% of total plasma kininase activity could not be explained by any of the enzymes tested suggests that there are still other kininases in rat plasma which remain to be discovered.

3-Mercaptopropionic Acid↗
PubMed1989

Kinins contribute to the contractile effects of rat glandular kallikrein on the isolated rat uterus.

Glandular kallikrein is known to promote contractions of the isolated, estrogenized rat uterus, perhaps independently of kinin formation. The recent availability of kinin receptor antagonists led us to study whether they might affect the oxytocic activity of kallikrein. DArg0-Hyp3-Thi5,8-DPhe7-bradykinin (8.5 x 10(-7) M) displaced the dose-response curves to both bradykinin (from 1.0 x 10(-9) to 4.0 x 10(-6) M) and kallikrein (from 4.7 x 10(-11) to 8.0 x 10(-9) M) approximately one order of magnitude to the right. This inhibition could not be due to a nonspecific effect on the uterine muscle, as the contractile response to oxytocin was not altered. In addition, carboxypeptidase B (a potent kininase) and kinin antibodies reduced the contractile response to kallikrein by 70 and 60%, respectively. Removal of the intervening agent restored the normal response. The effect of kallikrein depended on its enzymatic activity, inasmuch as kallikrein inactivated with D-Phe-Arg-Arg-CH2Cl was not oxytocic. Prolonged or multiple exposures to kallikrein completely abolished uterine response, whereas the effect of bradykinin was unaltered. Uterine horns rendered insensitive to kallikrein by prolonged exposure still contracted in response to trypsin. Kininogen was present in the uterine tissue in a concentration of 1.5 +/- 0.3 ng of bradykinin equivalents per mg wet wt. No more than 15.9 +/- 1.2% of this total was due to plasma contamination. Only 21.5 +/- 2.9% of total kininogen could be cleaved by kallikrein. We conclude that part of the oxytocic activity of kallikrein is related to generation of kinins from a kallikrein-sensitive kininogen present in the isolated rat uterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

A new kinin moiety in human plasma kininogens.

Recently, we isolated a new kinin from human urine and tentatively identified it as [Ala3]-Lys-bradykinin. However, there were inconsistencies between the properties of the naturally occurring new kinin and synthetic [Ala3]-Lys-bradykinin. In the present work, we determined whether the new kinin was released from human plasma kininogen, and further investigated the structure of the new kinin. After incubation of plasma (n = 6) with human urinary kallikrein, kinins were separated by HPLC and measured by RIA. The new kinin and Lys-bradykinin were found representing 23 +/- 3 and 76 +/- 6%, respectively, of total kinins released (2.0 +/- 0.4 micrograms/ml). The new kinin was also released from both purified low- and high-molecular-weight kininogens, representing 40-42% of total kinins released. Amino acid sequencing and composition analysis indicated that the structure of the new kinin was [Hyp3]-Lys-bradykinin (Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and not [Ala3]-Lys-bradykinin. We conclude that an important proportion of human kininogens contain hydroxyproline instead of proline in position three of the bradykinin moiety.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of a non-kallikrein arginine esterase from dog urine.

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.

Amino Acids↗
PubMed1988

The assay of glandular kallikrein and prekallikrein in human mixed saliva.

The measurement of glandular kallikrein in biological fluids most often utilizes a synthetic substrate, H-D-valylleucylarginine-p-nitroanilide (S-2266), which assesses amidase activity. Although this substrate has reasonable specificity for glandular kallikrein, other tryptic-like proteases found in mixed saliva may also cause hydrolysis. The primary purpose of this study was to assess the accuracy of the use of this substrate for the measurement of glandular kallikrein in human mixed saliva. An additional objective was to determine the presence of prekallikrein in mixed saliva. The addition of soybean trypsin inhibitor (SBTI), which inhibits other tryptic-like enzymes but not glandular kallikrein, resulted in an approx. 30 per cent decrease in the hydrolysis of S-2266 by centrifuged mixed human saliva. A correlation of 0.918 was obtained between the biological assays for kinin release and amidase activity in 19 subject samples. Amidase activity increased following treatment of saliva with trypsin, indicating the presence of prekallikrein in human mixed saliva. It is concluded that S-2266 is an accurate substrate for the assay of glandular kallikrein in human mixed saliva; that the inclusion of SBTI in the assay mixture is needed to inhibit non-kallikrein proteases that may also hydrolyse the synthetic substrate; and that prekallikrein is present in mixed saliva. Thus any future studies of changes in the level of kallikrein in saliva may wish to consider the presence of both active and total levels of glandular kallikrein.

Amidohydrolases↗