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Biomedical subjects

A S Brecher

Publications and source records attributed to A S Brecher.

At least 19 recordsLinked to original sources
PubMed1999

The effect of chlorambucil upon lysozyme activity.

Chicken egg white lysozyme is progressively inhibited by diazoacetyl-DL-norleucine methyl ester (DANME) and by chlorambucil at concentrations of 3.4 x 10(-3) M and 5 x 10(-3) M respectively over a three-hour time period. DANME inhibits lysozyme activity to the extent of 87%, and chlorambucil inhibits the enzyme to the extent of 93%. N,N',N"-triacetylchitotriose [(NAG)3], which binds to subsites A, B and C of the enzyme protects lysozyme from DANME inhibition to the extent of 40% of the total activity when added to the enzyme at a concentration of 3.6 x 10(-3) M prior to the addition of DANME. (NAG)3 protects the enzyme from inhibition by chlorambucil to the extent of 14% of the total activity when added to the enzyme at a concentration of 5.6 x 10(-3) M prior to the addition of chlorambucil. Since DANME reacts exclusively with carboxyl groups, and since aspartic acid 101 is required for binding the carbohydrate substrate at site A, it is suggested that (NAG)3 may bind reversibly to the active site of the enzyme, thereby protecting aspartic acid 101 from esterification by DANME and subsequent inactivation. Chlorambucil, which may react with carboxyl, amino, imidazole and thiol groups, more likely acts upon a larger number of susceptible sites, thereby causing irreversible alkylation and conformation changes. As a bifunctional alkylating agent, it may also cross-link with two available nucleophiles. The K(m) for lysozyme with M. lysodeikticus as a substrate in wholly aqueous medium was determined to be 0.05 mg/mL. The inhibitor exhibits a partially uncompetitive upon pre-incubation with the enzyme, and a mixed inhibition between competitive and noncompetitive when pre-incubated with the substrate.

Animals↗
PubMed1999

Coagulation protein function: the influence of acetaldehyde-modified heparin on thrombin activity.

BACKGROUND: The affect of acetaldehyde-treated heparin on thrombin activity has been investigated using factor II-deficient human plasma. METHODS: It was observed that 0.021 units of heparin exerts a marked inhibition of thrombin activity (1.03 units) as measured by clotting times, prolonging the clotting times from 9.6 +/- 0.1 seconds to 24.8 +/- 0.1 seconds. However, when the heparin is preincubated with 447 mmol/L acetaldehyde at RT for 30 minutes prior to mixing with thrombin, a clotting time in excess of 200 seconds is observed. Clotting times remain elevated with heparin-acetaldehyde mixtures of 89.4, 17.9, 3.6, and 0.72 mmol/L acetaldehyde, with corresponding clotting times of > 200, 156.0 +/- 2.1, 81.6 +/- 1.0, 38.8 +/- 0.6 seconds, respectively. At 140 mumol/L acetaldehyde-heparin mixtures, the clotting time was 17.0 +/- 2.0 seconds. RESULTS: These data support the hypothesis from this laboratory that acetaldehyde-modified heparin enhances coagulation time. They further indicate that thrombin is targeted by the acetaldehyde-treated heparin. Heparin-acetaldehyde mixtures also reacted with plasma prior to the addition of thrombin to modestly prolong coagulation time. Similarly, but more effectively, thrombin/heparin mixtures increased the clotting time of acetaldehyde-exposed plasma. These data further suggest the possibility that reactions of acetaldehyde and heparin are not restricted to those with thrombin, and that they may extend to other blood factors/proteins. CONCLUSIONS: The amount of heparin (0.021 units) required to substantially affect clotting time of thrombin (1.03 units) is substantially lower than that required to prolong clotting of 0.1 mL of whole plasma (0.36 units), by an order of magnitude. It is inferred that heparin may interact with numerous cationic proteins or proteins with cationic domains in blood plasma, among them being the clotting factors.

Acetaldehyde↗
PubMed1998

Coagulation protein function V: diminution of antithrombin III function by acetaldehyde.

The anticoagulant activity of antithrombin III (ATIII), as observed in a plasma-free system consisting of thrombin and fibrinogen, is readily reduced by acetaldehyde (AcH) at concentrations of 447, 89.4, and 17.9 mM. Whereas control thrombin-fibrinogen mixtures clotted in 17.7+/-0.75 sec, ATIII prolonged clotting time to 55.0+/-1.75 sec on preincubation with thrombin for 30 min at room temperature. On subsequent preincubation of ATIII with the AcH for 30 min at room temperature and passage of the mixture through Sephadex G-25 minicolumns to remove excess AcH, the eluates were tested for anticoagulant activity. Clotting times of 20.9+/-1.0, 32.3+/-1.0, and 45.3+/-1.6 sec were obtained with 447, 89.4, and 17.9 mM AcH-ATIII mixtures, respectively. These data suggest that functional groups on ATIII, such as guanidiniums, aminos, and others are susceptible to adduct formation with AcH, thereby altering the shape and charge of the anticoagulant. As a consequence of this type of reaction, an altered molecule of reduced biological activity may be produced. These experimental results may explain, in part, the reduction in ATIII levels reported by others in patients with alcoholic liver disease.

Acetaldehyde↗
PubMed1998

Acetaldehyde inhibits chymotrypsin and serum anti-chymotrypsin activity.

BACKGROUND: Chymotrypsin (CT) and CT-like enzymes contribute to the dynamics of metabolism by their participation in digestion, peptide hormone generation and catabolism, fertilization of ova and inhibition of thrombin-induced platelet aggregation, among other processes. The frequency of pancreatitis is observably higher in alcoholics, and pancreatic enzymes have been associated with localized vascular damage, thrombosis and pancreatic necrosis. METHODS: Since CT is a major pancreatic enzyme and may serve as a link between pancreatitis, coagulopathy, and alcoholism, the affect of acetaldehyde (AcH) the primary metabolite of ethanol, upon the enzyme and upon the influence of human serum thereon was studied. RESULTS: It was observed that CT activity upon glutaryl-L-phenylalanine-b-naphthylamide was inhibited to the extent of 23.7%, 52.5%, and 96.7% by 44.7, 89.4, and 447 mmol/L AcH in a fluorometric assay whereby the enzyme was dialyzed to remove excess AcH prior to assay. The p values were < 0.04. Aliquots of human serum (10 microL, 20 microL, 30 microL, 40 microL, 50 microL, and 100 microL) inhibited 40 micrograms of CT by 13%, 37.7%, 65.3%, 89.8%, and 92.8%, respectively (n = 6; p = < 0.05). The serum did not hydrolyze the fluorogenic substrate. On the other hand, AcH added to serum at 447, 224, 112, or 56 mmol/L resulted in 42.6%, 42.6%, 52.9%, and 60.3% inhibition of CT relative to a 69.1% inhibition of the enzyme by serum alone (n = 6; = p < 0.01). CONCLUSIONS: These data show that AcH clearly decreases the antichymotryptic activity of serum (consisting of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin). The incomplete inactivation of chymotrypsin by serum and partial inactivation of CT inhibitor(s) by AcH suggest the possibility that CT leaked into the circulation, (in alcoholic pancreatitis) may be available in blood to lower the clotting potential induced by thrombin-activated platelets, and that a greater amount of CT might be available in the blood of alcoholics, thereby contributing, in part, to the prolongation of clotting times.

Acetaldehyde↗
PubMed1995

Protection of chymotrypsin from inactivation by a N-mustard analog.

Chymotrypsin activity is rapidly inactivated by the N-mustard anti-tumor drug, chlorambucil. Since mustards react with thiols, amines, carboxyls, imidazoles, and sulfide sites on proteins, N-acetylcysteine, 2 proprietary protein hydrolyzates, beta-mercaptoethanol, ethanolamine, and sodium lactate were tested for their capacity to protect chymotrypsin from inactivation by the mustard. In each instance, protection was afforded to chymotrypsin. In as much as N-acetylcysteine protected chymotrypsin from inactivation by chlorambucil, it is suggested that this thiol compound may serve as a detoxication agent and may not require prior transformation into glutathione by cells in order to reduce mustard levels within the cells, as suggested by Smith and Gross (Proceedings of the NATO Panel VIII meeting, Grenoble, France, 1991.) It is further suggested that amino acids present as biosynthetic and degradative components of cells may detoxify mustards.

Amines↗
PubMed1994

Acetaldehyde alters coagulation protein function.

Acetaldehyde is the first metabolite of ethanol and has the potential to react with proteins and alter their function. This study evaluated the function of clotting proteins that had been preincubated with acetaldehyde as compared to those incubated with buffer or ethanol as controls. Thrombin, fibrinogen, thromboplastin, or whole plasma were preincubated with 1.8-447 mM acetaldehyde, 1.7-429 mM ethanol, or buffer for varying time periods prior to use in a clotting assay. Clot formation was measured with an automatic fibrometer. Acetaldehyde prolonged the clotting time but ethanol did not. These experiments indicate that circulating acetaldehyde would have the potential to react with proteins of the clotting system and alter their function. Therefore, it is possible that not all of the abnormalities in coagulation in alcohol abusers result from inadequate hepatic synthesis. Perhaps some of the deranged coagulation may be the result of the interaction of acetaldehyde with coagulation proteins.

Acetaldehyde↗
PubMed1992

Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli.

alpha-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the source of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine amino-peptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis.

Amino Acid Sequence↗
PubMed1989

Regulation of adrenal renin messenger ribonucleic acid by dietary sodium chloride.

Zona glomerulosa (ZG) and zona fasciculata (ZF/M) poly(A)+ RNA were isolated from the adrenals of bilaterally nephrectomized female Sprague-Dawley rats and hybridized to a full-length 32P-labeled 1423-base pair (bp) renin cDNA as well as a 698-bp renin cDNA KpnI segment (corresponding to amino acids 92-325) by the dot blot procedure using Bio-Rad Zeta Probe membranes. Extensive hybridization was observed with ZG mRNA, and only slight binding was seen with ZF/M mRNA. These results extend earlier reports from this laboratory indicating that the enzymic activity for renin is predominantly localized in ZG cells. Hence, high message levels account for the high enzymic activity. Adrenal ZG poly(A)+ RNA was also isolated from rats maintained on normal and sodium-deplete diets for 15 days and was hybridized to the radiolabeled 698-bp renin probe. Essentially twice the amount of probe was bound to the message from salt-deplete ZG tissue compared to message from normal ZG per microgram mRNA. Hybridization was proportional to the amount of poly(A)+ RNA employed over the range of 0-1 microgram, suggesting the applicability of this procedure for approximate quantitation purposes. The membranes were freed from the 32P-labeled renin cDNA and subsequently rehybridized with a 32P-radiolabeled 1200-bp beta-actin cDNA probe. It was observed that ZF/M poly(A)+ RNA contained more beta-actin message than ZG poly(A)+ RNA, indicating a greater transcription rate for beta-actin in ZF/M tissue in contrast to transcription of the renin gene.

Adrenal Glands↗
PubMed1978

Interaction of chlorambucil with tRNA.

Preincubation of purified mixed tRNAs from Escherichia coli K12-MO with 2.94 mM chlorambucil (CAB) for 2 h at 37 degrees C results in the inhibition of the capacity of mixed tRNAs to accept alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine by 100, 71, 100, 100, 100, 95, 32, 88, 36, 26, 96, 78, 44, 31, 34, 98, 38, and 17% respectively. Preincubation of tRNA with 0.75 mM and 0.29 mM CAB inhibited aminoacylation by aspartic acid to the extent of 69 and 17% respectively. CAB has no apparent effect upon the capacity of ATP to function in the formation of aminoacylated tRNALeu.

Adenosine Triphosphate↗
PubMed1977

Identification of alpha1-acid glycoprotein, alpha2-macroglobulin and antithrombin III as components of normal and malignant human tissues.

alpha1-Acid glycoprotein, alpha2-macroglobulin, and antithrombin III have been identified, by immunological means, as components of the 90000 X g supernatant fraction of malignant and adjacent normal human breast, colon, and anal tissues, as well as malignant stomach and ileum. Malignant lung tissue only contained alpha1-acid glycoprotein. These protease inhibitors are immunologically equivalent to those present in human plasma.

Antithrombins↗
PubMed1977

Characterization of the interaction between chymotrypsin and heparin.

Heparin forms a complex with chymotrypsin which is active towards glutaryl-L-phenylalanine-p-nitroanilide (GPANA) and glutaryl-L-phenylalanine-beta-naphthylamide (GPNA) at pH 7.6. The activity of chymotrypsin towards GPANA at pH 7.6 is enhanced in the presence of heparin. Heparin does not bind at the active site of the enzyme since proflavin is not displaced from the active site of chymotrypsin upon complex formation. The heparin-chymotrypsin complex migrates under basic polyacrylamide disc gel electrophoresis conditions to a position intermediate between heparin and free chymotrypsin. The complex is dissociable under acidic polyacrylamide gel electrophoresis conditions. It is estimated that one to three molecules of heparin can bind to each chymotrypsin molecule on the basis of electrophoretic and enzymic activity data.

Binding Sites↗
PubMed1976

The hydrolysis of biologically active peptides by bovine lung tissue factor (thromboplastin).

Tissue factor apoprotein and relipidated tissue factor preparations extensively hydrolyze bradykinin, Lys-bradykinin, Met-Lys-bradykinin, substance P, [Asp1, Ile5]-angiotensin II, [Asp1, Ile5]-angiotensin I, and human fibrinopeptide A while acting more slowly on [Sar1, Ile5]-angiotensin II, [Me2Gly1, Ile5]-angiotensin II, bradykinin potentiating pentapeptide from B. jararaca, luteinizing hormone-releasing hormone, melanocyte stimulating hormone-release-inhibiting factor (Pro-Leu-Gly-NH2), and oxytocin. No hydrolysis of thyrotropin-releasing factor or bradykinin potentiating nonapeptide from B. jararaca is observed. Relipidated and apoprotein tissue factor act at identical rates under the conditions of the assay. Dansylation and chromatography of tissue factor-peptide incubation mixtures further indicate that relipidated and apoprotein tissue factor also hydrolyze peptides by identical mechanisms. No fewer than six bonds are hydrolyzed in bradykinin while the angiotensins and substance P are degraded to constituent amino acids. Only the N-terminal alanine is released from fibrinopeptide A. 2-Mercaptoethanol greatly inhibits the hydrolysis of bradykinin by relipidated tissue factor.

Angiotensin II↗
PubMed1975

Isolation and identification of alpha1-antitrypsin as a component of normal and malignant human breast and other tissues.

Alpha1-Antitrypsin has been detected using radial immunodiffusion in the 90,000g supernatant fraction of malignant and adjacent normal human breast, colon, and anal tissue, as well as malignant lung, stomach, and ileum. Immobilized chymotrypsin, bound to Sepharose or Affi-Gel 10 has been utilized to separate 11 peaks of antiproteolytic activity by affinity chromatography of normal and malignant human breast tissue extracts. Glycoproteins are associated with eight of the peaks. Peak 1 contains predominantly alpha1-antitrypsin in addition to a minor component. The purification of the inhibitors, as judged by disc gel electrophoresis, is extensive. In some peaks, only one or two protein bands are observed, suggesting that affinity chromatography on Sepharose- or Affi-Gel 10-chymotrypsin might be used for the isolation of alpha1-antitrypsin and other inhibitors in preparative amounts.

Anal Canal↗