Progression and treatment of chronic adult periodontitis.

BACKGROUND: The periodontal status of 41 medically healthy adults with untreated chronic periodontitis was monitored before and after scaling and root planing (SRP). METHODS: During a 6-month pretreatment phase, clinical measurements, digital subtraction radiography (DSR) analysis of alveolar bone, and measurement of gingival crevicular fluid (GCF) prostaglandin E2 (PGE2) levels were undertaken. SRP was provided during a 1-month treatment phase. Clinical, radiographic, and biochemical analyses were repeated in a 6-month post-treatment healing period. RESULTS: Pretreatment: no clinically significant changes in mean plaque indices (PI), probing depths (PD), bleeding on probing (BOP), or relative clinical attachment levels (CAL) were detected (P>0.05). DSR revealed small but statistically significant bone height (0.04 mm) and mass (0.97 mg) loss (P<0.001). GCF PGE2 levels gradually increased from 38.8 ng/ml at month 1 to 79.4 ng/ml at month 6. Post-treatment: statistically and clinically significant reductions were observed in mean PI, BOP, and PD (P<0.05). A statistically significant reduction in CAL was noted (P<0.05). The trend towards progressive bone loss was halted and reversed, and a statistically significant decrease in GCF PGE2 concentrations was detected (P<0.001). Smokers, non-smokers, and ex-smokers did not differ significantly in PI, BOP, CAL, radiographic, or biochemical parameters at any time. Mean PD was significantly greater in current smokers than in non- and ex-smokers (P<0.005). PD reduced comparably in all 3 smoking subgroups following treatment (P<0.01). CONCLUSIONS: Conventional clinical measurements failed to identify disease progression over a 6-month period. Significant improvements were observed in clinical parameters after SRP, and a trend towards progressive bone loss was halted and reversed. Regular and frequent maintenance visits are important following treatment to maintain improvements in clinical parameters. Smokers had deeper probing depths than non- and ex-smokers, but pockets were reduced significantly and comparably in all 3 smoking subgroups following efficacious treatment.

Crevicular fluid prostaglandin E2 levels in periodontitis-resistant and periodontitis-susceptible adults.

The aim of this cross-sectional study was to determine concentrations of prostaglandin E2 (PGE2) in gingival crevicular fluid (GCF) in a cohort of periodontal disease-resistant (PDR) adults with chronic gingivitis but with minimal evidence of bone loss, and to compare these data with GCF-PGE2 levels in patients with untreated chronic adult periodontal disease (CAPD). 20 PDR and 35 CAPD subjects with mean (+/-se) ages 52.4 (+/-2.9) and 43.7 (+/-1.2) years respectively, were recruited. GCF was sampled from 6 sites in each PDR subject and 4 sites in each CAPD subject. The GCF-PGE2 concentrations were determined by enzyme immunoassay (Assay Designs). Whole mouth medians of site-specific GCF-PGE2 concentrations were calculated for each subject. The means of the median GCF-PGE2 concentrations were: PDR 54.94+/-4.06 ng/ml; CAPD 41.57+/-2.91 ng/ml (p=0.009). We hypothesise that the higher concentrations of PGE2 in the PDR group may be associated with the proliferating pocket epithelia of the chronic gingivitis. In the CAPD cohort, there were no differences in GCF-PGE2 concentrations between subgroups of smokers (n=13), ex-smokers (n=11) and non smokers (n=11). In the PDR cohort, 19/20 subjects were non-smokers.

Longitudinal changes in TCRB variable gene expression and markers of gingival inflammation in experimental gingivitis.

The aim of this study was to gain information of the cellular and molecular events which occur during the development of experimental gingivitis and to determine whether such changes occur in the presence or absence of alveolar bone resorption. Clinical, radiographic, biochemical and immunological variables were monitored in a 3-week, single-centre, experimental gingivitis study of 10 healthy volunteers. Following screening and professional prophylaxis to achieve visibly healthy gingival status, subjects abstained from all oral hygiene practises in one maxillary (test) quadrant for a period of 21 days. At days 0 and 21, in test and (contralateral) control quadrants, % bleeding on controlled pressure probing (% BOP) was calculated, and radiographic alveolar bone status was assessed using bilateral standardised vertical bite-wing radiographs and digital subtraction radiography (DSR) analysis. In test quadrants, gingival crevicular fluid (GCF) was sampled from 4 sites per subject with Periopaper strips, and prostaglandin E2 (PGE2) levels measured using an enzyme immunoassay (EIA) kit. At days 0, 7 and 21, one interdental papilla was surgically excised from the test quadrant, and the expression of T cell receptor B variable (TCRBV) genes was investigated using a reverse transcription-polymerase chain reaction (RT-PCR) procedure. At days 0, 7 and 21, peripheral blood lymphocytes (PBL) were isolated and additionally investigated for TCRBV gene expression. Following 21 days of plaque accumulation in test quadrants, a statistically significant increase in % BOP scores confirmed the presence of gingival inflammation (p<0.001). DSR analysis revealed that there were no significant alveolar bone changes in either the test or control quadrants between days 0 and 21 (p>0.05). EIA analysis of GCF samples identified a significant decrease in mean GCF PGE2 concentrations from day 0 to day 21 (p<0.05). RT-PCR analysis indicated that genes from all 3 TCRBV families studied (TCRBV-2, -6, -8) were expressed in the PBL samples at all time points and in healthy gingival tissues at day 0. A restriction in the expression pattern of TCRBV genes similar to those which have previously been reported in chronic periodontitis was noted at gingivitis sites. It is possible that such an event may identify susceptibility to periodontal disease independently of other positive predictive markers such as GCF-PGE2.

Effects of ketorolac tromethamine mouthrinse (0.1%) on crevicular fluid prostaglandin E2 concentrations in untreated chronic periodontitis.

The effects of topical ketorolac tromethamine mouthrinse (0.1%) on gingival crevicular fluid (GCF) prostaglandin E2 (PGE2) concentrations were investigated in a 6-week, randomized, double-blind, placebo-controlled, parallel group, single center study of 42 patients with moderately advanced chronic adult periodontitis. Following screening, GCF was sampled from 6 sites per subject with filter paper strips and PGE2 levels measured using an enzyme immunoassay kit. Only those subjects with mouth median GCF PGE2 concentrations >30 ng/ml entered the rinsing phase. Eligible subjects were allocated placebo rinse in the first 2-week period (days 0 through 14), either ketorolac rinse (test group, n = 21) or placebo rinse (control group, n = 21) in the second 2-week period (days 14 through 28), and placebo rinse in the third 2-week period (days 28 through 42). Full mouth median GCF PGE2 concentrations were calculated for each subject at days 0, 14, 28, and 42, and group means were compared. From day 0 to day 14, no significant changes in GCF PGE2 concentrations were detected in either study group (P > 0.05). Utilizing mean GCF PGE2 concentrations at days 0 and 14 as covariates, no significant differences were observed in adjusted mean PGE2 levels at days 28 and 42 between the study groups (ANCOVA, P > 0.05). A statistically significant increase in GCF PGE2 levels was noted at days 28 and 42 in the placebo group (P < 0.01), but not in the ketorolac group (P > 0.05), when compared to baseline, however. GCF PGE2 levels were further studied in a subset of volunteers (n = 11) during a 12-hour period following first rinsing with mouthrinse (active or placebo) at day 14. GCF was sampled 0, 2, 4, 6, 8, and 12 hours post-rinsing. Mean PGE2 levels were higher in the placebo subgroup than in the ketorolac subgroup, and increased gradually over the 12-hour period in both subgroups. These data indicate that 1) 14 days of rinsing with 0.1% ketorolac mouthrinse controlled the elevation of GCF PGE2 observed in the placebo group but did not actually reduce GCF PGE2 concentrations and 2) changes in GCF PGE2 levels were not detectable in the 12-hour period following first rinsing with ketorolac.

A 9.75-Mb map across the centromere of human chromosome 10.

We present a yeast artificial chromosome (YAC) and pulsed-field gel electrophoresis (PFGE) map across the centromere of human chromosome 10 that links expressed sequences in 10p11 to expressed sequences in 10q11.2. This map is the first of its kind to link genes across a human centromere. It consists of a 2.5-Mb YAC contig extending from 10p11 to our previously published 5.35-Mb PFGE map of the centromeric satellite arrays, and a 2.65-Mb YAC contig extending from these satellite arrays to 10q11.2. This map covers approximately 6.5-7% of the total DNA of chromosome 10. Two Généthon genetic markers, D10S578 and D10S604, are included. These markers are only 1 cM apart but are separated by a physical distance of more than 9.2 Mb, including the centromere. This gives a ratio of genetic to physical distance of 0.11 cM/Mb, 9-11 times lower than average estimates for the human genome and chromosome 10. Markers linked to the centromere include the duplicated zinc finger genes ZNF11A, ZNF33A, and ZNF37A (which map to 10p11) and ZNF11B, ZNF33B, and ZNF37B (which map to 10q11.2). Restriction mapping confirms that the genes on each arm lie in opposite orientation with respect to the centromere, consistent with the hypothesis that a pericentric inversion has occurred in this region during primate evolution.

Human brain leucyl aminopeptidase: isolation, characterization and specificity against some neuropeptides.

In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11.1) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-1M-1) but the peptides Tyr-Gly-Gly and Tyr-Gly-Gly-Phe-Leu (Leu5-enkephalin) were much better substrates (specificity constants 8300 and 18050s -1M-1 respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn++. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin 1-8, 1-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).

Purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle.

The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.

Characterization of aminopeptidases in human kidney soluble fraction.

Fractionation of human kidney soluble extract (with which the majority of cellular aminopeptidase activity is associated) via anion exchange chromatography resolved four types of separable aminopeptidase (relative activity in parenthesis): alanyl aminopeptidase (EC 3.4.11.14; 50%); arginyl aminopeptidase (EC 3.4.11.6; 30%); leucyl aminopeptidase (EC 3.4.11.1; 18%) and pyroglutamyl aminopeptidase (EC 3.4.19.3; 2%). The further purification (via gel filtration chromatography and preparative electrophoresis) and characterization of each aminopeptidase has been described; the aminopeptidase tissue profile for human kidney was found to be similar to that previously obtained for human skeletal muscle and brain tissue using a similar experimental approach, i.e. the same enzymes, with corresponding similar characteristics, are present in each tissue. These results suggest that soluble aminopeptidases may be of fundamental importance in general cell protein catabolism. Degradation of the following aminoacyl-AMC derivatives via kidney soluble extract has been shown to be due principally to hydrolysis by alanyl aminopeptidase: glutamyl, glycyl, isoleucyl, methionyl, ornithyl, phenylalanyl, prolyl, seryl, tyrosyl and valyl. We would suggest that measurement of the soluble extract-derived aminopeptidases described in this paper in urine may lead to an improvement upon existing assay procedures for early detection of kidney damage.

Specificity of action of human brain alanyl aminopeptidase on Leu-enkephalin and dynorphin-related peptides.

The major cystosolic aminopeptidase (alanylaminopeptidase) was purified to homogeneity from human cerebral cortex and the specificity of its actions on a series of Leu-enkephalin-related peptides of increasing chain length was determined. In each case, only the N-terminal Tyr-Gly bond was hydrolysed. Kinetic analysis of the data revealed that the specificity constant (kcat/Km;s-1M-1) falls with increasing chain length from a maximum of 13.6 x 10(4) for Leu-enkephalin (5 residues) to 5.8 x 10(2) for dynorphin (1-13). Dynorphin 1-17, while not being degraded itself acted as a competitive inhibitor (Ki = 2.7 microM) of the degradation of smaller peptides. Beta-endorphin was not hydrolysed by analylaminopeptidase, nor did it act as an inhibitor of the enzyme.

Comparison of major cortical aminopeptidase activity in normal brain and brain from patients with Alzheimer's disease.

In order to develop a greater understanding of the importance of peptide catabolism in the intracellular protein degradation process in normal and pathological human brain, we have undertaken a systematic investigation of the aminopeptidase group of enzymes. Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were hydrolysed by normal human cortical soluble extract, fractionation of the latter via anion exchange and gel filtration chromatography resolved only 4 separable aminopeptidase types (activity relative to alanyl aminopeptidase in parenthesis): alanyl (EC 3.4.11.14, 100%); arginyl (2 isoenzymes, EC 3.4.11.6, 15%); pyroglutamyl (EC 3.4.19.3, 4%); and leucyl (EC 3.4.11.1, 1%). Thus approx. 80% of the total soluble aminopeptidase activity in normal human cerebral cortex can be accounted for by a single enzyme, the major cortical aminopeptidase. The activity of this enzyme was measured in the soluble fraction prepared from 4 cortical regions (occipital, frontal, temporal and parietal) in a series of 8 patients with Alzheimer's disease and compared with corresponding data in 8 control normal patients. Although enzyme activity varied in these cortical regions, the activity in corresponding brain areas in the Alzheimer's disease and normal groups was very similar. These findings suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from altered activity of the major cortical aminopeptidase in the cortical tissue of patients with this disorder.

Rationalization of aminopeptidase activities in human skeletal muscle soluble extract.

Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were found to be hydrolysed by human skeletal muscle soluble fraction, fractionation of the latter via anion-exchange and gel-filtration chromatography resolved only five types of separable aminopeptidase (with activity relative to alanyl aminopeptidase in parentheses): alanyl aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.14, 100%), arginyl aminopeptidase (two isoenzymes, L-arginyl-L-lysyl)-peptide hydrolase, EC 3.4.11.6, 15%); pyroglutamyl aminopeptidase (5-oxoprolyl-peptide hydrolase, EC 3.4.19.3, 3%); leucyl aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1, 1.5%) and alpha-glutamyl aminopeptidase (0.2%). Thus over 80% of the total aminopeptidase activity (expressed in relative terms) in human skeletal muscle soluble fraction can be accounted for by a single enzyme, the major aminopeptidase. A single peak of activity, which co-eluted with the major aminopeptidase after anion-exchange and gel-filtration chromatography, was obtained after assay with the following aminoacyl-7-amino-4-methylcoumarin derivatives: glycyl-, isoleucyl-, lysyl-, methionyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. Thus, the hydrolysis of these derivatives by skeletal muscle soluble fraction occurs principally via the major aminopeptidase and not by specific enzymes, as previously suggested (Wada and Aoyagi, 1983). These results illustrate the difficulty in measuring individual aminopeptidase activities in muscle homogenate and soluble fraction, and the danger in ascribing apparent aminopeptidase activity to 'specific' enzymes.

Purification and characterization of two soluble Cl(-)-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).

Serum creatine kinase-BB levels and cerebral cortical creatine kinase activity in senile dementia of the Alzheimer type.

A rise in serum creatine kinase-BB (CK-BB) levels has been reported previously in cases of dementia. In the present study the levels of serum CK-BB have been measured in patients clinically assessed to have senile dementia of the Alzheimer's type (SDAT) and in cognitively intact individuals, matched for age, by a specific two-site monoclonal immunoradiometric assay. No significant difference was found between the 2 groups. Total creatine kinase activity in temporal cortex (Brodmann area 21 and 22) was also found to be similar in brains from SDAT or control cases, obtained at autopsy. These results suggest no major change in the permeability of the blood-brain barrier to this enzyme in SDAT patients.

Absence of biochemical heterogeneity in McArdle's disease. A high resolution SDS-polyacrylamide gel electrophoresis study.

Muscle biopsy extracts from a series of 6 patients with McArdle's disease were investigated by analytical SDS-polyacrylamide gel electrophoresis, to establish the presence or absence of the myophosphorylase protein subunit. In 4 cases, the band corresponding to the myophosphorylase subunit was totally absent from the electrophoretic staining pattern, and in 2 cases was present, but with a greatly reduced staining intensity compared with control normal patients; thus in none of the cases of McArdle's disease investigated was there evidence for a myophosphorylase subunit band of comparable staining intensity to that found in control normal patients. This result contrasts with previously reported findings (Feit and Brooke 1976) which suggested that McArdle's disease exists in biochemically heterogeneous forms; in one form of the disease myophosphorylase being totally absent and in a second form present to a similar extent as normal, but in an inactive form. On the basis of the results reported in this paper, we would suggest that myophosphorylase deficiency is a single gene disorder characterized by the absence or marked reduction of the myophosphorylase protein.

Purification and characterization of a neuropeptide-degrading aminopeptidase from human brain.

The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.

Specificity of neuropeptide degradation by two calcium-activated neutral proteases from human skeletal muscle.

Two calcium-activated neutral proteases (CAPI & II) were purified from human skeletal muscle by anion exchange, gel filtration and affinity (antipain-Sepharose and Blue Ultrogel A4R) chromatography. The enzymes were homogenous as judged by polyacrylamide gel electrophoresis, and have similar properties with the exception of the Ca2+ concentration required for optimum activity (CAP I = 0.1 mM; CAP II = 1 mM). Both enzymes hydrolysed a wide variety of neuropeptides. In six cases, the products were separated and identified by hplc and amino acid analysis. Neurotensin was hydrolysed at Tyr3-Glu4; dynorphin1-13 at Arg8-Arg9; LH-RH at Gly6-Leu7; CCK-8 at Phe8-NH2, substance-P at Met10-NH2; somatostatin at Thr10-Phe11. Although differences in the rates of neuropeptide degradation were noted for the two CAP's the specificity was the same for these six peptides. It is suggested that conformational requirements may be more important than side chains adjacent to the cleavage site in directing the specificity of CAP.

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle.

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.