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Our purpose was to evaluate a two-stage newborn hearing screening program using automated auditory brainstem response (AABR) before discharge and to describe our follow-up program. This study used 4085 infants born in the Seirei-Hamamatsu and Mikatahara General Hospitals during a 2-year period. The initial screening test was performed 2 or 3 days after birth at an intensity of 35 dBnHL. For the infants who were referred from this test, the re-screening test was performed 5 or 6 days after birth. Diagnostic work-up with auditory brainstem response (ABR), otoacoustic emissions (OAE), and a conditioned orientation reflex audiometry (COR) test were performed by the age of 3-6 months. The referral rate was 1.20% (49/4085 infants) in the first test and 0.71% (29/4085 infants) in the two-stage screening. The two-stage screening procedure was able to reduce the false-positive rate from 0.83 to 0.34%. The incidence of bilateral and unilateral congenital hearing loss diagnosed by ABR was 8/4085 (0.20%) infants and 7/4085 (0.17%) infants, respectively. One infant with congenital cytomegalovirus infection, who passed the two-stage AABR tests, was diagnosed with hearing loss 1 month after birth, using ABR. The two-stage measurement of AABR is effective and time efficient due to significant decreases in the referral rate and the false-positive rate.

This study aimed to examine molecular mechanisms responsible for the metabolic fate of S-nitrosoglutathione (GSNO) in endothelial cells. After addition of 1 mM GSNO in culture medium, concentration of S-nitrosothiols (RSNO) significantly decreased with concomitant accumulation of nitrite (NO2-) only in the presence of human endothelial cells (ECV304), while no change in RSNO decomposition and NO2- accumulation was observed in case of S-nitrosocysteine. Bathocuproine disulfonic acid (BCS), a chelator for Cu(I), prevented the cell-mediated decomposition of RSNO and accumulation of NO2-. Chelator for Cu(III), Fe(II), or Fe(III); inhibitors of gamma-glutamyltranspeptidase; or a superoxide quenching enzyme had no effect on the cell-mediated degradation of RSNO and accumulation of NO2-. These results indicate that cellular Cu(I) would play a major role in the conversion of GSNO into NO2-. We recently demonstrated that human glyoxalase I (Glo I) interacts with GSNO in vitro and within cells. When Glo I interacts with GSNO, Glo I is inactivated and is chemically modified with pI alteration on 2D gels. So, we examined effect of Cu(I) chelation on the Glo I response. As a result, chelation of cellular Cu(I) by BCS enhanced the inactivation and chemical modification of Glo I by GSNO. The Glo I response could be detected when the cells were exposed to GSNO at 10 microM, corresponding to the concentration of RSNO under physiological conditions, with pretreatment of BCS. Metal chelators for copper and iron ions had no effect on the sensitivity of Glo I to an nitric oxide (NO) radical donor. These results indicate that chelation of cellular Cu(I) potentiates the sensitivity of GIo I to GSNO. The observation in the present study implicates that intracellular levels of GSNO might be elevated, accompanying with stabilization of extracellular RSNO.

Glutathione peroxidase (GPx) activity was detected in the ascite fluid of rats injected intraperitoneally with 2.5% heat-denatured casein solution. Activity in the ascite fluid increased with time after the injection of casein, and reached a maximum at 24 h. The active component was concentrated with successive 35% ammonium sulfate precipitation and Activated Thiol-Sepharose 4B column chromatography from the ascite fluid of rats at 24 h after the injection of casein. No N-terminal amino acid of the protein corresponding to GPx was detected by automatic amino acid sequence analysis following separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a polyvinyl difluoride (PVDF) membrane. Following BrCN treatment of the protein, the N-terminal amino acid sequences of two 14 and 2.6 kDa peptide fragments were found to be S-G-T-I-Y-E-Y-G-A-L and K-I-H-D-I-R-W-N-F-E, respectively. The former and the latter fragments corresponded to sequences beginning at the 37th and 176th amino acid residues of rat extracellular GPx (eGPx), respectively. The exclusive presence of eGPx in the ascite fluid of rats elicited by casein was confirmed immunologically by ELISA, immuno-precipitation and Western blotting assays. No other GPx isozymes such as cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx) or intestinal GPx (iGPx) were detected. eGPx activity and protein were also detected in the pleuritic fluid of rats following injection of 2% carrageenan. These findings indicate that eGPx appears at various sites of acute inflammation in rats. This pattern is due to leakage from circulation as a result of the increased capillary permeability at inflammation sites elicited by chemotactic factors.

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we examined the NO-responsive proteins in cultured human endothelial cells by two-dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NORPs)] with different pI values responded to NO donors. One NORP (pI 5.2) appeared in response to NO, whereas another (pI 5.0) disappeared. These proteins were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4. 4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in response to NO, and all NO donors tested inhibited its activity in a dose-dependent manner. Activity and normal electrophoretic mobility were restored by dithiothreitol and by the removal of sources of NO from the culture medium. Glox I was selectively inactivated by NO; compounds that induce oxidative stress (H(2)O(2), paraquat and arsenite) failed to inhibit this enzyme. Our results suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-sensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.

The cytosol of rabbit lung alveolar macrophages contains a high amount of peroxidase, which reduces 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) in the presence of glutathione. This peroxidase was purified 69-fold to homogeneity with overall recovery of activity of 18.5%. The molecular mass of the enzyme was approximately 80 kDa by gel filtration, and emerged as a single band at 23.1 kDa under reducing condition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amino-terminal sequence of the purified peroxidase was completely identical to the sequence deduced from cellular glutathione peroxidase (cGPx) gene of rabbit liver. No other activity that reduces 5-HPETE to 5-HETE was observed during purification. These results suggest that cGPx plays an important role in metabolism of lipid hydroperoxides, especially HPETE, in lung alveolar macrophages.

Diabetes was induced in rats by the intravenous administration of streptozotocin (STZ; 60 mg/ml). The activity of cholesterol esterase (CEase) in various tissues was determined by use of either a fluorogenic or radioactive substrate. Significant CEase activity was detected in extracts of pancreas. The specific activity of pancreatic CEase was considerably greater in pancreatic extracts from diabetic rats compared with normal rats. The activity of pancreatic CEase increased 6 d after the injection of STZ, but the difference was not statistically significant. It reached a maximum value approximately twice that of normal pancreas at 30 d with statistic significance. The highest specific activity of pancreatic CEase was found in the cytosolic fraction from diabetic rats, whereas the specific activity of the enzyme was lowest the same fraction from normal rats.

The subcellular distribution of the elastase in human myeloid leukemia HL-60 cells was studied in comparison with that in normal leukocytes. On differential centrifugation, most of the elastase activity of HL-60 cell lysates was recovered in the 105,000 x g supernatant, while that of human peripheral blood leukocyte lysates was recovered in the 500 x g precipitate (azurophil granule-rich fraction). Moreover, on Percoll density gradient centrifugation, the elastase activity in HL-60 cell extracts was recovered in the lightest fraction with none in the azurophil granule-rich fractions, whereas most of the activity in leukocyte extracts was recovered in the azurophil granule-rich fractions. This subcellular localization of elastase did not change when HL-60 cells differentiated into monocytes and granulocytes by induction with 12-O-tetradecanoyl phorbol-13-acetate and retinoic acid, respectively. Furthermore, on Sephadex G-75 gel filtration, the elastase activity in HL-60 cell extracts was eluted earlier than that in leukocyte extracts. The size estimation indicated that the elastase of HL-60 cells was 36-30 kDa, corresponding to the size of an elastase precursor reported. The relevance of a large form of the elastase in HL-60 cells to its subcellular localization is discussed.

A proteinase extracted with 1M NaCl from particulate fraction of the postnuclear fraction of mouse myeloid leukemia M1 cells was partially purified by Bio-Gel HTP treatment and Sephadex G-75 gel filtration. The apparent molecular mass of the proteinase was 26,000 Da and the isoelectric point was about pH 10. The enzyme activity was inhibited by phenylmethanesulfonylfluoride, chymostatin, and soy-bean trypsin inhibitor. It hydrolysed specifically Suc-Ala2-Pro-Phe-4-methylcoumaryl-4-amide (MCA). NaCl and KCl enhanced several times the activity for Suc-Ala2-Pro-Phe-MCA, but not that for fluorescein-labeled albumin and fibrinogen. These enzymic properties of the major proteinase are similar to those of chymotrypsin and cathepsin G. The role of a cathepsin G-like proteinase in relation to M1 cell differentiation is discussed.

Amidolytic activity of thrombin for Boc-Val-Pro-Arg-MCA was inhibited by CaCl2 at relatively high concentrations. Kinetic analysis showed neither competitive nor noncompetitive inhibition by CaCl2 of thrombin. The nonspecific interaction of thrombin with calcium ions prolonged fibrinogen-clotting and neutralization by antithrombin III (AT III) in the absence of heparin. However, calcium ions had no effects on rate of thrombin inactivation in dilute solution and affinity of the enzyme for Sepharose 6B. The inverse effects of calcium ions on the thrombin activities in vitro and on generation of the enzyme in plasma ex vivo depended mostly on the ion concentration.

Thrombin-clotting time of fibrinogen was delayed by inositol hexasulfate (IHS). Clotting time of the recalcified plasma was also delayed by IHS. Moreover, when IHS was intravenously injected into anesthetized male rabbits (0.11 mmoles/kg), it delayed clotting time of the recalcified plasma. IHS prevented generations of factor Xa and thrombin in the recalcified plasma. It is probable that IHS inhibits the clot-formation of the recalcified plasma in competition with calcium ions.

Kinetic analyses were made of the thrombin/antithrombin III (ATIII) reaction in the presence of catalytic amounts of three kinds of mammalian HA-heparin (bovine, pig and whale), which have the same affinities for ATIII. In the absence of NaCl, the first-order rate constant was higher with whale HA-heparin than with the other two HA-heparins. However, the strength of the interactions of thrombin with the HA-heparins was in the order, bovine greater than pig greater than whale. Thus, the slow reactions in the case of bovine and pig HA-heparins were probably due to preferential binding of the two HA-heparins to thrombin rather than to ATIII, thus causing the HA-heparins to be inhibitory for the reaction by reducing the turnover rates of the polysaccharides as catalysts. In the presence of 0.15M NaCl, the first-order rate constants were slightly higher in the case of pig HA-heparin than of the other two HA-heparins. Since the strength of the interactions of these HA-heparins with thrombin was in the order, pig greater than or equal to bovine greater than whale, the faster reactions were probably due to higher associations of the enzyme with the essential HA-heparin-ATIII complex.

Less than a stoichiometric amount of bovine chymotrypsin was progressively inactivated by a bovine AT III preparation. The rate of inactivation was not influenced by a synthetic substrate. Since chymotrypsin has low affinity for heparin, the polysaccharide only doubled the inactivation of the enzyme by the AT III preparation. At a low salt concentration, about two-thirds of the chymotrypsin was instantaneously inactivated by the AT III preparation in the presence of heparin. These observations indicate that inactivation of chymotrypsin is due to AT III itself, not to another contaminating inhibitor(s).

Inactivation of thrombin was enhanced by dextran sulfate (DS) coated on polypropylene tubes with plastic adhesive. Long interaction of thrombin with DS was not essential to enhance the enzyme inactivation. Albumin inhibited the inactivation of thrombin by DS-coated tubes, but had no effect after interaction of the enzyme with DS. NaCl, especially, sodium ions, inhibited the inactivation by DS-coated tubes. Enzyme inactivation occurred at 37 degrees C, but not at 4 degrees C. Thus, the inactivation was due not only to ionic interaction with DS, but also to intramolecular perturbation of thrombin.

Heparin with low affinity (LA-heparin) to antithrombin III (AT III) enhanced the rate of inactivation of thrombin by AT III. The enhancement of the rate was saturable with AT III and was proportional to the LA-heparin concentration. Although the rate-enhancement in the presence of LA-heparin decreased with increase in NaC1 concentration, it was comparable with that in the presence of high affinity-heparin (HA-heparin) in the absence of NaC1. Inactivation of thrombin by AT III in the presence of dextran sulfate (DS) was also sensitive to NaC1 concentration. These findings indicate that free AT III is favorable for binding to the complexes of thrombin and highly sulfated polysaccharides having low affinities to AT III in the absence of NaC1.

The stereospecificity of peptidyl dipeptide hydrolase [EC 3.4.15.1] was investigated. Six free and N-blocked alanyl peptides containing D-alanine were synthesized and tested as substrates. Their susceptibilities were determined by measuring Ala-Ala release by cation exchange column chromatography. Their Michaelis constants, Km values, and velocity maxima, Vmax values, were also determined. The enzyme showed high stereospecificity for an amino acyl residue in position 3 from C-terminus: it had an absolute requirement for the alanyl residue of the L-configuration in this position. An alanyl residue of the L-configuration in position 1 or 2 increased, but was not essential for activity. The enzyme showed little stereospecificity for an alanyl residue in position 4 from the C-terminus.

A pico-molar concentration of thrombin was significantly inactivated by antithrombin III (AT III). The pseudo-first order rate constant, k'app, and the second order rate constant, k", were inversely proportional to the concentration of thrombin. The k'app and k" values increased and decreased, respectively, with increase in the concentration of AT III. All of these results indicated that the rate of the thrombin/AT III reaction was determined with the molar ratio of the two rather than their absolute concentrations.

Commercially available heparin preparations slightly enhanced the rate of thrombin/antithrombin (AT) III reaction at pH 6.05 in the absence of NaCl. However, this accelerative activity was significantly lower than that induced by heparin with high affinity for AT III (HA-heparin), probably due to the formation of the binary complexes of HA-heparin-AT III as well as that composed of thrombin and heparin with low affinity for AT III (LA-heparin). The HA-heparin-catalyzed thrombin/AT III reaction was faster in the presence of 0.1 M NaCl at pH 6.05 than that in the absence of the salt. LA-heparin and dextran sulfate (DS) were also found to accelerate the thrombin/AT III reaction rate, but neither substance catalyzed the formation of the complex in the presence of 0.1 M NaCl at pH 7.4. LA-heparin was also confirmed to compete with HA-heparin for enhancement of the thrombin/AT III reaction. Thus, it appears that AT III tends to form a ternary complex with the thrombin-DS or thrombin-LA-heparin complex, even in the presence of 0.1 M NaCl, whereas factor Xa reacts with the AT III-DS or AT III-LA-heparin complex. These results indicate that HA-heparin is the only substance having the ability to catalyze the thrombin/AT III reaction, and that its turnover rate is markedly elevated in the presence of strongly electropositive and electronegative ions because of the decreased affinity of the enzyme for heparin under such conditions.

Egg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid. Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.

Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor.

To study molecular-mass dependence of dextran sulfate (DS) for interactions with several plasma proteins, a commercial preparation of the sulfated polysaccharide was fractionated by gel filtration chromatography into six subfractions with relatively different molecular masses. Simple two-component systems were available to measure the interactions of the proteins with the subfractions of DS. These were done to determine the rates of time-dependent changes in intrinsic fluorescence of thrombin and fibrinogen, and the enzyme inactivation in the presence of DS. Their interactions were also confirmed in three-component systems, in which the interactions of DS with thrombin and fibrinogen were measured by the displaced binding by FTC-heparin, and DS-enhanced proteolysis by chymotrypsin, respectively. Moreover, the neutralization of factor Xa by antithrombin III (AT III) depended on the molecular mass of DS. All the results obtained indicate that most of the general interactions of thrombin, fibrinogen, and probably AT III increased with increasing molecular mass of DS.

The effects of pH on salt stimulation of the rates of hydrolysis of three substrates by human leukocyte elastase were studied. The enzyme was most active at pH 10.5, 8.0-8.5, and 9.5 for the hydrolyses of fluorescein isothiocyanate-labeled S-carboxymethylated bovine serum albumin (FITC-CM-BSA), succinyl-L-Ala-L-Pro-L-Ala-7-methylcoumaryl-4-amide (Suc-APA-MCA), and succinyl-L-Ala3-p-nitroanilide (Suc-Ala3-pNA), respectively, in the absence of NaCl. The enzyme was activated by 0.5 M NaCl similarly at all pHs tested for the hydrolysis of Suc-Ala3-pNA, but more at neutral and alkaline pH values, respectively, for the hydrolyses of FITC-CM-BSA and Suc-APA-MCA. Thus, in the presence of 0.5 M NaCl, the enzyme was most active at pH 8.0 and 10.0 with FITC-CM-BSA and Suc-APA-MCA, respectively. In contrast, the proteolytic activity of porcine pancreatic elastase was somewhat inhibited by 0.5 M NaCl.

Although loss of chymotrypsin activity in dilute solution deviated from first order kinetics at low enzyme concentration, it displayed first order kinetics at concentrations more than 4 nM. First order rate constants varied with the ratio of surface area to volume, with the kind of vessel containing the enzyme, and with the particular test material (DS, polybrene, lecithin or BSA) coating the vessel. The reaction was saturable at lower chymotrypsin concentrations in glass than in polypropylene tubes, while less enzyme was lost at high concentrations. All these facts showed that loss of enzyme activity is incompletely, but markedly, due to a solid surface-catalysed reaction. Intrinsic fluorescence of native chymotrypsin at pH values 8 and 3, and of active site-blocked enzyme, decreased with time at 37 degrees C. No extensive autolysis of chymotrypsin was observed during the time-dependent loss of enzyme activity. Therefore, the apparent loss of chymotrypsin activity in dilute solution was mainly due to an irreversible conformational change of the molecules, as associated with the solid-surface-catalysed reaction.

Peptidyldipeptide hydrolase [angiotensin I-converting enzyme, EC 3.4.15.1] was inhibited by inorganic and organic phosphorus compounds tested, except for beta-glycerophosphate, 5'-AMP, and 5'-ADP, at the reagent concentrations used. Orthophosphate and pyrophosphate nonspecifically inhibited the enzyme activity. The enzyme was also inhibited specifically by carboxylates. The degree of inhibition by aliphatic monocarboxylates increased in proportion to their chain length up to C14. Aromatic and omega-phenylalkylcarboxylates also inhibited the enzyme activity. The enzyme was noncompetitively inhibited by acetate, 3-phenylpropionate and laurate. The Ki's for acetate, 3-phenylpropionate, and laurate were 60, 3.3, and 2.5 mM, respectively.

The activity of carboxypeptidase A [EC 3.4.12.2] was inhibited by 3-phenylpropionate derivatives (p-aminocinnamate, 3-p-aminophenylpropionate and 3-p-acetylaminophenylpropionate), and to investigate its use as a ligand for affinity chromatography 3-p-aminophenylpropionate was directely and indirectly coupled to Sepharose 4B. carboxypeptidase A was adsorbed only on 3-p-aminophenylpropionate bound to the gel through p-phenylenediamine as a spacer. Carboxypeptidase A from pancreas was purified by a combination of this affinity adsorbent and ion exchange chromatography. The purified carboxypeptidase A had a homogeneity similar to that of a commercial product, as judged by disc gel electrophoresis. The carboxypeptidase activity of Pronase was slightly retarded on the gel column, but could not be separated from its caseinolytic activity. Angiotensin I-converting enzyme [peptidyl dipeptidy hydrolase, EC 3.4.15.1] obtained from hog kidney cortex was not bound to the gel.

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].

Amidolytic activity of thrombin increased proportionally with NaCl concentrations up to 1 M NaCl, due to an increase in the turnover rate with no change in the Km of the enzyme. Neutralization of thrombin by antithrombin III (AT III) was maximum in 0.1 to 0.15 M NaCl, but completely absent at high NaCl concentrations. Moreover, ratio of rates of the amidolytic activity of thrombin to of the enzyme/AT III reaction was not constant at several pHs. The rate of the thrombin/AT III reaction appeared to be inversely correlated with the enzyme's stability. Therefore, it was concluded that some factor(s) other than the active site of the thrombin molecule was more important in its primary interaction with AT III.

The interaction of bovine pancreatic alpha-chymotrypsin with dimyristoyl phosphatidylcholine (PC) vesicles was measured turbimetrically. The protein interacted with the vesicles at NaCl concentrations of above 0.8 M. The turbidity reached a plateau on increase in the amount of either the protein or the vesicles in the presence of a fixed amount of the other component. The precipitates formed contained both PC and protein in ratios varying with the initial amount of each component. On mixing chymotrypsin and PC vesicles, time-dependent turbidity increase was high at below pH 2.5, but relatively small at neutral and alkaline pH values. Apolar interaction between the two components was confirmed by demonstrating an increase in fluorescence intensity of chymotrypsin in the presence of the PC in 1 M NaCl. The turbidity of a mixture of PC vesicles and bovine serum albumin (BSA) increased even in the absence of 1 M NaCl, whereas the turbidities of mixtures of the vesicles and lysozyme or alpha-lactalbumin did not change with time in the presence of 1 M NaCl at pH 8.0.

The rate of the thrombin/antithrombin III (AT III) reaction was decreased in the presence of free polybrene or protamine. The reaction rate was also decreased in protamine-coated tubes and tubes on which polybrene was absorbed nonspecifically. The reaction was also prevented when either thrombin or AT III was transferred into noncoated tubes after instantaneous contact with protamine-coated tubes or tubes with polybrene. These facts suggest that the thrombin/AT III reaction rate is determined with concentrations of reactive protein species.

Dextran sulfate and polybrene were immobilized on polypropylene tubes with plastic adhesive. Serum albumin was also immobilized on the tube by heat-denaturation. In the those tubes thrombin was protected from inactivation in the presence of 0.1 M NaCl remarkably. Cholesterol coated on the tube also protected the enzyme from inactivation. The results indicate that amounts of thrombin protected by these four coating materials corresponded to the amount of thrombin inactivated on surface of the tube. At least half of the thrombin was also inactivated within 20 sec at 37 degrees C in the presence of quartz sand, supporting a significant contribution of a solid surface to temperature-dependent inactivation of enzyme in dilute solution.

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin. Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme. These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.

Proteolysis of fibrinogen by bovine trypsin and chymotrypsin was enhanced by heparin, dextran sulfate (DS) and polyvinyl sulfate (PVS) in the presence of 0.1 M NaCl. Decrease in intrinsic fluorescence of fibrinogen with time was also enhanced by DS and PVS in the absence of NaCl, but not in the presence of 0.1 M NaCl. Thus, increase in susceptibility of fibrinogen to proteases in the presence of three polysulfate was more sensitive than time-dependent conformational change of the substrate protein.

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.

Clostridial collagenase (EC 3.4.24.3) catalyzes the hydrolysis of (Pro-Pro-Gly)5 at minimum of three different rates, producing Pro-Pro, Gly-Pro-Pro and Gly-Pro-Pro-Gly, and various intermediate peptides. The intermediate and final products were separated by cation-exchange column chromatogrphy and identified, and their rates of formation were measured. Pro-Pro was released most rapidly with formation of the tridecapeptide. After the initial release of the N-terminal Pro-Pro, hexa- and heptapeptides were formed in larger amounts than tri-, tetra-, nona- and decapeptides from the tridecapeptide. The rates of disappearance of the intermediates decreased in the order trideca- greater than deca- and nona- greater than heptapeptide. The results indicate that the enzyme hydrolyzes inner linkages of the tridecapeptide having N- and C-terminal Gly residues, forming large peptides, preferentially to outer linkages, forming the tri- and tetrapeptides.

At high thrombin concentration, loss of enzyme activity was proportional to the initial enzyme concentration. At low thrombin concentration, loss of activity followed first order kinetics, indicating mainly denaturation (inactivation) of the enzyme. Dextran sulfate stimulated thrombin denaturation, while serum albumin and polyethylene glycol 6000 (PEG) protected the enzyme from denaturation. These results indicate that the enzyme molecule is stabilized by weak interactions with albumin and PEG, but destabilized by strong interaction with dextran sulfate.

Of four Tris-salts tested (chloride, sulfate, phosphate and acetate), chloride caused complete elution of antithrombin III (AT III) from a heparin-Sepharose column and sulfate caused partial elution. AT III was also partially eluted from the column with sodium acetate, but not Tris acetate. On the other hand, thrombin was eluted from the column with the Tris-salts in the order chloride greater than sulfate greater than acetate, but was not eluted with Tris-phosphate. Thrombin was also eluted from the column with sodium acetate. These findings indicate that the affinity of heparin to AT III was influenced only by strongly electronegative ions, whereas its affinity to thrombin was affected by both strongly electropositive and strongly electronegative ions.

Dextran sulfate (DS) had a greater ability to elute thrombin adsorbed on a small Sepharose 6B column than heparin did, while chondroitin sulfate A had little ability. It is probable that the strength of the interaction of thrombin depends mostly on the charge-density of strongly acidic sulfate groups in the polysaccharides. The change in intrinsic fluorescence intensity of thrombin with time was closely correlated with the rate of inactivation of the enzyme in the presence of sulfated polysaccharides. Both rates were affected by the pH of the solution in the presence of the polyanions. The rates in the presence of DS were highest at pH 6.05 among the three pHs tested, while they were enhanced only at pH 6.05 by heparin, but not by chondroitin sulfate A. Therefore, extensive charge-neutralization of thrombin by the sulfated polysaccharides is able to induce time- and temperature-dependent intramolecular conformational change (irreversible denaturation) of the enzyme molecules.

Dextran sulfate (DS)-enhanced inactivation of alpha-thrombin was faster at pH 6 than at pH 8 in the absence of NaCl. Substrate protected only partly thrombin from inactivation by DS at pH 6. Some amidolytic activity of alpha-thrombin inactivated by DS was restored time-dependently by the addition of NaCl. Moreover, beta and gamma-thrombin, and other intermediates were not formed from alpha-thrombin with DS-enhanced inactivation. All these results indicate that autolysis of alpha-thrombin in dilute solution is not responsible for enhancement of reversible inactivation of alpha-thrombin by DS.