Proteolytic profile of Treponema vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity

The proteolytic profile of Treponema vincentii ATCC 35580 was studied using PZ-PLGPA (phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-argi-nine; a substrate of bacterial collagenases) and amino acid 2-naphthylamides (2NA) as substrates. The cell extracts showed high activity toward PZ-PLGPA, Na -L-arginyl-2NA and N γ-L-glutamyl-2NA. Gel permeation chromatography revealed 2 major endopeptidases (I and II) hydrolyzing PZ-PLGPA, the molecular weights of which were 75,000 and 23,000, respectively. Enzyme I was stable enough for subsequent fast protein liquid chromatography on an anion exchange column. The enzyme had a broad pH optimum of 6.5 to 7.5 with PZ-PLGPA as substrate, hydrolyzed gelatin and was moderately inhibited by metal chelators, but was very sensitive to p -chloromercuribenzoic acid (PCMB). Enzyme II with a pH optimum of 7 to 8 was more labile, quite sensitive to PCMB and moderately inhibited by chelators. A high-molecular weight arginine aminopeptidase (mol. wt. > 200,000) was sensitive to PCMB and showed a value of 0.55 mM for K_m in the hydrolysis of Na -L-arginyl-2NA. The hydrolysis of PZ-PLGPA and gelatin suggests that this organism may contain collagenolytic proteinases. Because the insoluble proteinase substrate Azocoll was not hydrolyzed, these enzymes may be active on soluble collagenous substances only. T. vincentii ATCC 35580 typifies an organism rich in PZ-PLGPA-endopeptidase, arginine aminopeptidase and γ-glutamylpeptidase activity.     

Properties of UDPG pyrophosphorylase from rat salivary glands and liver.

UDPG pyrophosphorylase (UTP: α-d-glucose-1-phosphate uridylyltransferase, E.C. 2.7.7.9) isolated from rat sublingual and submandibular glands, is similar to that from liver and is located in cytoplasm (80 per cent) and in the nuclear fraction (20 per cent). DEAE-cellulose chromatography of the extracts isolated two forms of the enzyme from liver but only one from the salivary glands. The uridylyltransferase from all 3 tissues showed high specificity for UTP as substrate. The enzyme activity was inhibited by the two reaction products, PP_i and UDPG. Mg²⁺ and Mn²⁺ enhanced the enzyme activity, whereas Ni²⁺ and Cd²⁺ inhibited it. Co²⁺ did not affect the enzyme from liver, caused 2-fold enhancement of the activity of the enzyme from the sublingual gland and inhibited the submandibular gland enzyme. The differences in the effect of cobalt ion can be taken advantage of for differentiation of the enzymes. The molecular weight of the enzyme from all 3 tissues was about 300,000.     

Effects of iron and ascorbic acid on acid phosphatases of the enamel organ of rat molars

Protein extracts from 6- to 11-day-old rat enamel organs were applied to columns of carboxymethyl-52 cellulose. Protein eluted from the columns was assayed for acid phosphatase activity with substrates para-nitrophenylphosphate (p-NPP). beta-glycerolphosphate (β-GP), ATP and phosphocasein. A weakly-bound peak of activity (A) emerged first which was insensitive to stimulation by iron and ascorbic acid. This enzyme hydrolysed only the phosphomonoester substrates (p-NPP and β-GP). A strongly bound peak of activity (B) emerged later and was completely separated from the first activity. It hydrolysed all substrates except β-GP, and was stimulated at least 10-fold by 0.1 mM ferrous ion (Fe²⁺) in the presence of a strong reducing agent (l.0mM ascorbic acid). Both substances were more effective as stimulators when used together than they were when each was used separately. Dependency on these co-factors for the development of full activity increased with purification, especially when phosphocasein was substrate. The results were similar for each age of rat used. These properties of enzyme B are parallel with those of the acid phosphoprotein phosphatases of liver and spleen, and the tartrate-resistant acid phosphatase of rat bone. We conclude that enzyme B requires iron and a reducing agent for full activity and has properties that distinguishes it from the classical acid phosphatases (E.C. 3.1.3.2.).     

Characterization of N-CBz-glycyl-glycyl-arginyl peptidase and glycyl-prolyl peptidase of Bacteroides gingivalis

Bacteroides gingivalis produces large amounts of proteolytic enzymes which may play a role in its virulence. These enzymes may participate in the tissue destruction of the inflammatory process. In this study, the characteristics of two such enzymes, N-CBz-glycyl-glycyl-arginyl peptidase (N-CBz-Gly-Gly-Arg peptidase) and glycyl-prolyl peptidase (Gly-Pro peptidase) were investigated. The enzymes eluted in different peaks from an anion exchange column. N-CBz-Gly-Gly-Arg peptidase was associated with cells up to 48 h in culture. If cultured longer, it also released in the supernatant. It exhibited optimal activity between pH of 7.0 and 7.5 and was readily inactivated by heat treatment (45°C for 15 min). The enzyme activity was inhibited by p-chloromercuribenzoic acid (PCMB), leupeptin and antipain, suggesting that it is a thiol protease. The B. gingivalis N-CBz-Gly-Gly-Arg peptidase was different from the serum enzyme that digests the same substrate. The serum enzyme was more resistant to heat treatment and was inhibited by diisopro-pylfluorophosphate (DFP). B. gingivalis also produced Gly-Pro peptidase that is released in the supernatant. The enzyme has an optimal pH range between 7.5 and 8.0. The B. gingivalis Gly-Pro peptidase was inhibited by DFP, suggesting that it represents a serine protease. The serum Gly-Pro peptidase did not differ from the bacterial enzyme with respect to its sensitivity to inhibitors; however, they were markedly different in heat sensitivity. The bacterial enzyme was completely inactivated at 60°C for 30 min, whereas the serum enzyme was not inactivated even at 1 h at 60°C.     

Phospholipase A2 in rat gingival tissue

Phospholipase A2 (PLA2) is a proinflammatory enzyme in the synovial fluids of all--and sera of some--patients with rheumatoid arthritis. Due to the similarities in pathogenesis between rheumatoid arthritis and periodontitis, we sought to study the enzymatic properties of PLA2 in periodontal tissue. In this study, we demonstrated PLA2 activity in rat gingival tissue, about 80% of which was present in the cytosolic fraction. We characterized the cytosolic PLA2 enzyme with respect to substrate specificity, sensitivity to detergent, Ca2+ ion dependency and optimum pH. We found that phosphatidylethanolamine, rather than phosphatidylcholine, was the preferred substrate, the Ca2+ ion was essential for the expression of PLA2 activity, the enzyme was active over a broad pH range, with the optimum at pH 9.0, and sodium-deoxycholate inhibited the enzyme activity strongly in a concentration-dependent manner. These results are consistent with those which have been obtained with synovial fluid PLA2 and suggest that gingival PLA2 may be involved in the pathogenic processes of gingivitis and periodontitis.     

Effect of xylitol-, sucrose-, and water-rinses on the composition of human palatine gland secretions

The chemical composition of stimulated human palatine secretions (HPS) was studied after rinsing with aqueous solutions of sucrose or xylitol, or with water only. Stimulation with water was associated with increased concentration levels of protein. The levels of sialic acids (per mg protein) and the specific arylamidase activities were simultaneously increased, within the physiologic range. In contrast to a previous report, these results indicate that HPS does contain arylaminopeptidases the activity of which is strongly increased by Cl-. The Cl(-)-dependent activity was revealed when using N-L-arginyl-2-naphthylamide as substrate. The filter paper method used is recommended for collecting HPS for chemical analyses. In spite of the use of a relatively effective and versatile stimulation procedure, only one third of the subjects produced detectable HPS. Very poor secretion of HPS should perhaps be considered in subjects wearing total dentures.     

A comparison of the alkaline phosphatases of rat dental pulp, bone, kidney, liver and intestine

The sensitivity of the dental pulp enzyme to heat was similar to that of enzymes from bone and kidney; alkaline phosphatases from liver and intestine were more stable to heat. L-Homoarginine strongly inhibited the enzyme activities from pulp, bone, kidney and liver but did not affect intestinal enzyme activity. Increasing the molarity of carbonate buffer or glycine buffer in the assay solution decreased intestinal alkaline phosphatase activity more markedly than enzyme activities of other tissues. In 100 mM glycine-NaOH buffer, the effects of Zn2+, Mg2+ and Ca2+ on pulp alkaline phosphatase activity were similar to those on the enzyme activities of bone, kidney and liver. The electrophoretic pattern of the pulp enzyme on sodium dodecyl sulphate-gels was identical with that of bone enzyme and differed from the patterns of enzymes from other tissues. These results suggest that the dental pulp alkaline phosphatase may be the same as bone enzyme.     

Effect of xylitol-, sucrose-, and water-rinses on the composition of human palatine gland secretions

Abstract – The chemical composition of stimulated human palatine secretions (HPS) was studied after rinsing with aqueous solutions of sucrose or xylitol, or with water only. Stimulation with water was associated with increased concentration levels of protein. The levels of sialic acids (per mg protein) and the specific arylamidase activities were simultaneously increased, within the physiologic range. In contrast to a previous report, these results indicate that HPS does contain arylaminopeptidases the activity of which is strongly increased by Cl⁻, The Cl⁻ -dependent activity was revealed when using N -L-arginyl-2-naphthylamide as substrate. The filter paper method used is recommended for collecting HPS for chemical analyses. In spite of the use of a relatively effective and versatile stimulation procedure, only one third of the subjects produced detectable HPS, Very poor secretion of HPS should perhaps be considered in subjects wearing total dentures.     

Hydrolytic and depolymerising enzyme activity of Prevotella intermedia and Prevotella nigrescens

OBJECTIVE: Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.
MATERIALS AND METHODS Nine strains of P. intermedio and 12 strains of P. nigrescens were studied. Lipolytic. saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromo-genic substrate methods.
RESULTS: All strains hydrolysed gelatine, casein. DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563^T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia , hydrolysed benzyl-arg-p-nitroanilide. All strains hydrolysed alkaline pnitrophenolphosphate (except P. intermedia DAL 100). produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-p-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.
CONCLUSIONS: P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspec-ies activity is variable.     

Studies on Dog Saliva III. Observations on Enzymes Acting on Ester Bonds and Glycosyl Compounds

Hydrolases acting on ester bonds and glycosyl compounds were studied in the supernatant and sediment fraction of centrifuged dog saliva. When comparing the values obtained to those of man the most interesting deviations were: (1) the lack of a-amylase in dog saliva. (2) the low activity in dog saliva towards 6-bromo-2-naphthyl-α-D-glucoside and glucuronide, (3) the low activity towards 1-naphthyl phosphate at the acidic pH in dog saliva, and (4) the lack of enzymes hydrolyzing 6-bromo-2-naphthyl sulphate in the dog saliva supernatant fraction. The enzymes of centrifuged dog saliva and its sediment were fractionated with Sephadex G-200 and DEAE-cellulose columns. DEAE-cellulose chromatography brought out three enzyme peaks with phosphomonoester hydrolase activity, one of which was active at a wide pH range, one at the acidic side and one at the alkaline side. Two enzyme peaks hydrolyzing 6-bromo-2-naphthyl sulphate were obtained with DEAE-cellulose chromatography from the sediment fraction of dog saliva. The hydrolysis of carboxylic acid esters was observed in several enzyme peaks both from the supernatant and sediment fractions. The chromatographic results resembled those obtained with man.     

Relationship of inorganic pyrophosphatase and para-nitrophenylphosphatase activities of alkaline phosphatase in the microsomal fraction of isolated odontoblasts

Some properties of inorganic pyrophosphatase (PPiase EC 3.6.1.1.) and para-nitrophenylphosphatase (p-NPPase EC 3.1.3.1) in the microsomal fraction of odontoblasts were investigated. The ratio of Mg2+:p-NPP and Mg2+:PPi for optimal enzyme activities was 1:1. A mutual substrate competition for PPiase and p-NPPase was described. In the presence of 0.1 mM EDTA, Mg2+ alone was not able to reactivate p-NPPase or PPiase. Instead, Zn2+ and Co2+ reactivated the PPiase, indicating they might act as cofactors for the enzyme. Mg2+ increased the PPiase activity, probably because Mg PP2-i was the true substrate for the enzyme. The diphosphonates ethane-1-hydroxy 1,1 diphosphonate (EHDP), methane diphosphonate (MDP) and dichloromethane diphosphonate (Cl2MDP) inhibited the PPiase activity.     

Observations on endopeptidases in human carious dentin

abstract – The enzymatic hydrolysis of hemoglobin (Hb) and N-α-benzoyl-DL-arginine-2-naphthylamine (BANA) was observed both in sound- and in carious human dentin. The enzyme activity expressed per mg protein in the sample and per min was at least ten times higher in carious than in sound dentin, as was also the DNA content in the samples. The hydrolysis of Hb and BANA was high over a relatively wide pH range, the optimum pH being 5.5 with Hb, and 7.0 with BANA, as substrates. EDTA, cetylpyridinium chloride and Hg²⁺ ions inhibited strongly the hydrolysis of BANA at pH 7.0 but only slightly at pH 10.4. The origin of these endopeptidase-like enzymes was thought to be mainly microbial. These enzymes were thought to hydrolaze proteins of the organic stroma of dentin during the carious process.     

Studies concerning the glucosyltransferase of Streptococcus sanguis

We have shown in previous studies that the glucosyltransferase (Gtf) enzymes of Streptococcus mutans have distinct properties when adsorbed to a surface. In the present study, we compared the activity of Gtf from Streptococcus sanguis, designated GtfSs, in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads, and determined the ability of its product glucan to support the adherence of oral microorganisms. Gtf from S. sanguis 804 NCTC 10904 was purified from culture supernatant fluids by means of hydroxyapatite chromatography. Enzyme and the substrate were prepared in buffers at pH values from 3.5 to 7.5. Maximum activity of GtfSs occurred between pH 5.5 and pH 6.5, whether in solution or adsorbed onto a surface. The solubilized and insolubilized enzymes showed highest activity at 40 degrees C; activity was reduced by 50(+/-2)% at 20 and 30 degrees C. The enzyme did not form glucans in either phase at 10 or 60 degrees C. The K(m), determined from Lineweaver-Burk plots, for the enzyme in solution was 4.3(+/-0.4) mmol/l sucrose, and the K(m) for the enzyme on sHA beads was 5.0(+/-1.0) mmol/l sucrose. The ability of the GtfSs glucan synthesized on the surface of sHA beads to support the adherence of oral bacteria was investigated. (3)H-thymidine-labeled bacteria (S. mutans GS-5, S. sobrinus 6715, S. sobrinus 6716, S. sanguis 10904, Actinomyces viscosus OMZ105E, A. viscosus 2085, and A. viscosus 2086) were incubated with sHA beads coated with GtfSs glucan. S. mutans GS-5 displayed the highest level of binding numerically. These results show that the GtfSs of S. sanguis is active on sHA beads, that the pH optimum for activity on a surface differs slightly from that in solution, and that its product glucan can support the adherence of oral microorganisms.     

Effects of fluoride on rat dental enamel matrix proteinases

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10(6) fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 microM fluoride was compared by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 microM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 microM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.     

Neuropeptide enzyme hydrolysis in human saliva

The possible hydrolysis of neuropeptides by human saliva was studied using leucine enkephalin as a model. The data obtained indicate that in the presence of saliva this substrate is partially hydrolysed, and that its disappearance corresponds to the appearance of peptides whose composition is consistent with that of the substrate hydrolysis by-products. The formation of these peptides indicates the presence of all three classes of enzymes known to hydrolyse enkephalins in other tissues: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. The activity of these enzymes appears to be altered by the presence of low molecular-weight substances, whose inhibitory activity is apparent on all three classes of enkephalin-degrading enzymes. Substrate degradation was higher in male than female saliva; these differences appear to be caused by lower activity of the enzymes, and higher activity of the low molecular-weight inhibitors, measurable in female as compared to male saliva.     

ATP-ase activity in the odontoblastic layer of rat incisor Determination with a radiochemical and a colorimetric method

The ATP-splitting enzyme activity in odontoblasts isolated from rat incisors has been studied by means of a radiochemical and a colorimetric micromethod. The results with the two methods were virtually identical. The reaction was linear with time for at least 45 min. The pH optimum was found to be 9.8 independently of the ATP concentration. Maximal substrate saturation occurred at a total ATP concentration of 3 mM. Ca²⁺ and Mg²⁺ ions activated ATP degradation. F- ions did not affect the activity at low concentrations, whereas higher concentrations were inhibitory. Na⁺ and K⁺ ions had no influence on ATP splitting enzyme activity, while PO4^3- ions were slightly inhibitory. Urea inhibited the enzyme activity at concentrations above 1.5 M, while EDTA and EGTA were strong inhibitors at very low concentrations. When incubating in the presence of low concentrations of specific inhibitors for nonspecific alkaline phosphatase, levamisole and R 8231, about 20% ATP degrading enzyme activity remained. In conclusion it is suggested that there are at least two ATP degrading phosphatases active at alkaline pH.     

Trypsin-like, chymotrypsin-like and glycylprolyl dipeptidase activities in gingival crevicular fluid from human periodontal sites with gingivitis

These activities were measured simultaneously, using synthetic fluorescent protease substrates, in gingival crevicular fluid collected at 6 pre-determined sites from 10 individuals with mild to moderate gingivitis. The three enzyme activities were detected in 85, 18 and 93% of the sites, respectively. The volume of fluid collected from discrete sites was significantly correlated with the total amount of substrate hydrolysed, but not with the specific rate of substrate hydrolysis. Log10 (total trypsin-like activity) was significantly correlated with the Gingival Index, Plaque Index and probing depth (r = 0.319, 0.423 and 0.336), while total glycylprolyl dipeptidase activity was significantly correlated with probing depth (r = 0.381). These findings add to knowledge of the biochemistry of gingival crevicular fluid, but the usefulness of such assays for diagnostic or monitoring purposes in periodontal diseases needs to be determined.     

Processing of acidic proline-rich proprotein by human salivary gland convertase

Previously it was found that proproteins for basic and glycosylated salivary proline-rich proteins (PRP) were cleaved prior to secretion from cells by furin, a well-known convertase. In contrast proproteins for acidic PRPs are not cleaved by furin or other convertases. To investigate the convertase responsible for in vivo processing of acidic PRP proproteins, homogenates of human sublingual glands were fractionated by centrifugation at 10,000 x g and 100,000 x g and activity demonstrated in all fractions. The 100,000 x g pellet was fractionated into Golgi, smooth endoplasmic reticulum and microsomal fractions with the latter containing the enzyme. Subfractionation of the microsomes revealed that the activity was located in the membrane proteins. Since the microsomes contain components of the secretory pathway the enzyme in this fraction may be responsible for intracellular cleavage of the acidic PRP proprotein. The enzyme was active at alkaline pH. It was strongly inhibited by metal chelators indicating that it is a metalloprotease. It was not inhibited by an acid protease inhibitor, but partly inhibited by some serine protease inhibitors indicating that serine proteases may play a role in degradation. Co2+ and to some extent Zn2+ activated the enzyme, but it was strongly inhibited by Hg2+ and Cu2+ as well as the organomercurial p-chloromercuribenzenesulfonic acid. Thus it appears that the enzyme contains an important -SH group. These characteristics indicate that the convertase is related to a group of metal- and thiol-dependent proteases known as thimet oligopeptidases, but in contrast to the latter enzymes the sublingual convertase was not inhibited by angiotensin antagonists.     

Differences in acidogenicity of S. sobrinus and S. rattus are linked to the catalytic efficiency of the glycolytic key enzyme phosphofructokinase

This contribution describes the biochemical properties of two catalytically different phosphofructokinases (PFKs) purified from Streptococcus rattus LB 2 (PFK-rat) and Streptococcus sobrinus OMZ 65 (PFK-sob), respectively. Steady-state kinetics revealed K(M) = 0. 8 mM for PFK-rat and K(M) = 0.08 mM for PFK-sob for F-6-P as the substrate. The enzymes also differ in their pH profiles: whereas the highest activity of PFK-rat was measured at pH = 8.0, the optimum pH of PFK-sob was at pH = 7.0. In addition, compared to PFK-sob, PFK-rat was more sensitive against the allosteric inhibitor ATP. PFK catalyzes a committed step of glycolysis, the main acid producing catabolic pathway. Thus, the catalytically more efficient enzyme isolated from S. sobrinus OMZ 65, especially at low pH, could explain the comparably high acidogenicity of this strain.     

Multiple forms of acid phosphatase in rat secretory enamel organ

Abstract – Acid phosphatase activity was studied biochemically in homogenates of secretory enamel organs from the rat. Incubations with crude homogenate failed to show distinct pH optima or kinetics characteristic for single enzymes. Crude homogenate activity was strongly inhibited by concentrations higher than 1 mM of NaF and Na-tartrate, and higher than 10 mM of ZnSO₄ and p -bromotetramisole oxalate. lOmM MgCl₂ gave a slight stimulation. CaCl₂, KCl and EDTA were uneffective. Electrophoretic separation of the crude homogenate acid phosphatase on Triton X-100 containing polyacrylamide gel demonstrated the presence of atleast three multiple forms of the enzyme. Two of them showed distinct pH optima at pH 4.4. The third one showed a broad pH plateau in the acid pH range. Kinetic studies of the three forms indicated single enzyme reactions. Two forms had electrophoretic mobilities similar to alkaline phosphatase. One form could be solubilized only after Triton X-100 treatment. All forms were strongly sensitive to 10 mM NaF when added to the reaction mixture. The sensibility to 10 mM ZnSO₄, CuSO₄, Na-tartrate and p -bromotetramisole oxalate differed between the different forms.