Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the major protein components of the virion: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently removed HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA₂ 185–211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a major site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM β-mercaptoethanol than in the presence of 50 mM. However, the complete removal of ectodomains of HA by the high-and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein M1. The action of trans-epoxysuccinyl-L-leucylamido)(4-guanidino)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl₂ On the hydrolysis of proteins HA and M1 by bromelain was investigated.     

The specificity of proteinases from Streptomyces griseus (pronase)

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A(2) contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-l-arginine and hippuryl-l-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.     

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.     

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.     

Topological location of diamine oxidase in the transverse plane of rabbit liver microsomal membranes

The transverse topology of diamine oxidase within rabbit liver microsomal membranes was studied by examining the proteolytic digestion of sealed or detergent-permeabilized microsomal vesicles. Trypsin and pronase had no effect on diamine oxidase activity in any incubation conditions tested, while nagarse treatment reduced by 60-70% the enzymic activity of intact microsomes; no further loss of activity was observed in the presence of detergent. These results demonstrate that the active site of diamine oxidase faces the cytoplasmic membrane surface, and suggest that it does not possess or expose on either membrane surfaces bonds susceptible to the proteolytic attack by trypsin or pronase. The possible significance and the biological implications of the results are discussed.     

Effect of SH-reagents on aldehyde dehydrogenase activity of the rat liver

SH-reagents: tetraethylthiuram disulphide (TETD), 5,5'-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (p-ChMB), N-ethylmaleimide (NEM) were studied for their effect on the aldehyde dehydrogenase activity of mitochondrion (isoenzymes I and II) and microsome (isoenzyme II) fractions of the rat liver. TETD is established to inhibit isoenzyme I and isoenzyme II activity of mitochondrial aldehyde dehydrogenase by 100 and 50%, respectively, and the microsomal enzyme activity by 20%. DTNB and NEM inhibit 30-50% of the activity in two isoforms of mitochondrial aldehyde dehydrogenase having no effect on the enzymic activity in microsomes; p-ChMB inhibits completely the activity of the enzyme under study both in the mitochondrial and microsomal fractions. A conclusion is drawn that SH-groups are very essential for manifestation of the catalytic activity in the NAD+-dependent aldehyde dehydrogenase from mitochondrial and microsomal fractions.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Increased susceptibility of carbamylated glutamate dehydrogenase to proteolysis

Glutamate dehydrogenase is very susceptible to carbamylation which results in loss of activity. The effect of a number of proteolytic enzymes (pronase, trypsin and chymotrypsin) on native and carbamylated glutamate dehydrogenase was tested. In all cases, the carbamylated enzyme was at least twice as susceptible to proteolysis as the native enzyme. Antibodies were prepared against glutamate dehydrogenase and carbamylated glutamate dehydrogenase; the carbamylated enzyme was antigenically indistinguishable from the native enzyme. Preliminary experiments indicate that the carbamylated glutamate dehydrogenase is taken up by ascites tumor cells while glutamate dehydrogenase is not. It seems possible that the effects described can be extrapolated to degradation by lysosomes and to other covalently modified enzymes.     

Immunohistochemical localization of glandular kallikrein in the endocrine and exocrine human pancreas

Fab fragments from two new monospecific anti-human tissue kallikrein sera were examined for their capacity to inhibit the functional activities of purified human urinary kallikrein and purified human pancreatic kallikrein. Fragments from a new anti-urinary kallikrein serum and from an anti-pancreatic kallikrein serum yielded mixed inhibition of kinin-generating activity and minimal inhibition of esterolytic activity. In contrast to the previously described "active site directed" anti-urinary kallikrein, these new antisera demonstrated little specificity for epitopes near the enzymatic site of urinary or pancreatic kallikrein. When used to localize kallikrein antigen in human pancreas obtained at surgery, IgG fractions of the new anti-kallikrein sera yielded moderate acinar and ductal staining in the absence of pretreatment of the tissue with trypsin or pronase. Short incubation with 0.125 mg/ml of either enzyme permitted the discrete localization of islet beta cell kallikrein antigen, while increased pronase concentrations decreased kallikrein antigen in both islets and exocrine tissue and led to islet destruction. Both antibody specificity and tissue preparation influence kallikrein localization in human pancreas.     

Properties of 5'-nucleotidase in rat heart sarcolemma

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.     

Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans.

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.     

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.     

Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.     

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.     

Covalent modification of the catalytic sites of the H+-ATPase from chloroplasts and 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.     

Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin.

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating an active site portion of the enzyme, prostaglandin synthetase. In the current study, the site of acetylation has been demonstrated to be a seryl residue at the NH2 terminus of the enzyme. Purified [3H]acetyl enzyme was prepared from seminal vesicle homogenates treated with [acetyl-3H]aspirin. The [3H]acetate to protein bond was stable to hydroxylamine, indicating an N-acetyl linkage. The [3H]acetyl enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. The 3H material isolated from the pronase digest was identified as N-acetylserine. This finding indicates that the oxygenase portion of prostaglandin synthetase has an NH2-terminal serine which is involved in enzymatic activity and is susceptible to acetylation by aspirin.     

Tryptic digestion of NADH dehydrogenase from alkalophilic Bacillus

The alkalophile NADH dehydrogenase (NADH: 2,6-dichlorophenolindophenol oxidoreductase) [EC 1.6.99.3] consists of two identical subunits of 65 kDa, and each subunit contains the catalytic and liposome-binding regions. On treatment with trypsin, the polypeptide exhibiting the liposome-binding property in one of the subunits was digested to form an enzymatically active hetero-dimer (40 and 65 kDa), and then the polypeptide in the other subunit was digested to form an active homo-dimer (40 and 40 kDa). The hetero-dimer bound to liposomes, but the homo-dimer did not. Kinetic analysis showed that removal of one or two of the polypeptides in the enzyme slightly affects its kinetic parameters. For all the enzyme species, NAD inhibited competitively with respect to NADH and non-competitively with respect to 2,6-dichlorophenolindophenol. The partially determined amino acid sequence of this alkalophile enzyme suggested that (i) a long random-coiled peptide (58 amino acid residues) or a portion of the peptide is located between the polypeptides with liposome-binding and catalytic properties, (ii) the polypeptide exhibiting liposome-binding property is in the amino terminal region of the enzyme, (iii) the amino acid sequences around the subtilisin and trypsin cleavage sites of the peptide are hydrophilic and on the surface of the protein molecule and therefore are susceptible to digestion, and (iv) the FAD-binding site is located near the amino terminal region of the catalytic region.     

Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.