Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases

The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine

Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.     

A new approach to the synthesis of branched and branched cyclic oligoribonucleotides.

The six-step synthesis of the di-triethylammonium salt of 5[prime]-O -trityl-6-N-pivaloyladenosine-2[prime]-(H -phosphonate)-3'-[(2-chlorophenyl) phosphate]9 from 3', 5'- O -(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-6-N-pivaloyla denosine1in 68% overall yield is described. Compound9is converted into a branched pentaribonucleoside tetraphosphate 24 and a branched cyclic pentaribonucleotide ('lariat') 25 by solution phase triester chemistry involving both H-phosphonate and conventional phosphotriester coupling reactions. The monomeric building block 9 is proposed as a universal synthon for the preparation of branched and branched cyclic oligoribonucleotides derived from adenosine.     

Amino acid sequence of human D of the alternative complement pathway

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.     

Study on highly diastereoselective synthesis of (2'R)-2'-deoxy[2'-2H]guanosine.

To develop an efficient method for the synthesis of a highly diasteroselective (2'R)-2'-deoxy[2'-2H]guanosine (1), studies of organic chemical conversion from 2'-bromo-2'-deoxy-N2-Isobutyryl-3',5'-O-TIPDS-guanosine (2) to 1 and a biological transdeoxyribofuranosylation of (2'R > 98% de)-2'-deoxy[2'-2H]uridine (4) were carried out. As the results, a highly diastereoselective synthesis of 1 was achieved by a biological transdeoxyribofuranosylation between 2,6-diaminopurine and 4 by the use of Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. The results will be described in detail.     

Syntheses and structure-activity relationships of novel, potent, and selective trans-2-[3-oxospiro[isobenzofuran-1(3H),1'-cyclohexan]-4'-yl]benzimidazole NPY Y5 receptor antagonists

Syntheses and structure-activity relationships of a novel class of 2-[3-oxospiro[isobenzofuran-1(3H),1'-cyclohexan]-4'-yl]benzimidazole NPY Y5 receptor antagonists are described. Optimization of the lead compound 2a by incorporating substituents into the 5-position or into both the 5- and 6-positions of the benzimidazole core part led to the identification of 5-(5-methyl-1,2,4-oxadiazol-2-yl)benzimidazole (2r: IC(50)=3.3 nM) and 5-(2-methyltetrazol-5-yl)benzimidazole (2u: IC(50)=5.9 nM), both of which are potent, selective, and orally bioavailable Y5 receptor antagonists.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

1-[3-Aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa

Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki=30 pM) with a rapid onset of inhibition (2.7x10(7) M-1 s-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.     

Novel synthesis of cholesterol analogs: condensation of pregnenolone with dihydropyran or dihydrofuran.

Pregnenolone 3-(2'-tetrahydropyranyl) ether (1) was condensed with 3,4-[2H]dihydropyran to mainly give (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (20R-3), according to nuclear magnetic resonance (NMR). Cold, dilute HCl in ethanol removed the tetrahydropyranyl group at C-3 and also opened the dihydropyranyl ring at the C-20 position of 20R-3 to give (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol (20R-5). Analogous results were obtained by condensing pregnenolone 3-acetate with 3,4-[2H]dihydropyran to provide (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-acetate (20R-4). Acid-catalyzed opening of the dihydropyranyl ring at C-20 in 20R-4 yielded 20R-7, which, on acetylation followed by crystallization, provided (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol 3,26-diacetate (20R-8), identical to the diacetate made from 20R-5. Varying the reaction sequence beginning with 20(R,S)-4 gave an 84:16 ratio of 20R to 20S in a mixture of 20(R,S)-8, according to NMR analysis. Crystallization of the mixture from methanol provided pure 20R-8. Condensing 2,3-dihydrofuran and 1 for producing (20R)-[5'-(2',3'-dihydrofuranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (6) gave instead (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol 3-(2'-tetrahydropyranyl) ether (20R-9) by partial hydrolysis during workup. Treating 20R-9 briefly with dilute HCl produced (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol (20R-10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and Evaluation of Serine Enzymes by [3H]-DFP Affinity Labeling in Spinach Plants

Serine enzymes were detected in spinach plants by affinity labelingwith [³H]-di-isopropyl phosphorofluoridate (DFP) and SDS-polyacrylamidegel electrophoresis. Two serine enzymes were detected in thedry seeds, and another 4 major and 3 minor serine enzymes weredetected in 48-hr soaked seeds, especially in the cotyledons.Fourteen serine enzymes, including 9 enzymes in the cotyledons,were detected in mature leaves. [³H]-DFP binding with some serineenzymes in mature leaves was inhibited by a prior treatmentwith phenylmethylsulfonyl fluoride, a more specific probe ofserine proteases. Other affinity labeling reagents for serineproteases, L-1-tosylamide-2-phenylethyl chloromethyl ketoneand N-p-tosyl-L-lysine chloromethyl ketone also decreased DFP-bindingto some serine enzymes. These results are evidence that the enzymes found are serineproteases. Natural inhibitors for serine proteases, leupeptin,aprotinin and soybean trypsin inhibitor had no effect on [³H]-DFPbinding. DFP-binding with all the serine enzymes detected inthe mature leaves was decreased by p-chloromercuric benzoatebut not by EDTA. (Received June 12, 1982; Accepted September 28, 1982)     

Antifungal activity of organobismuth compounds against the yeast Saccharomyces cerevisiae: structure-activity relationship

Antifungal activity of organobismuth(III) and (V) compounds 1-9 was examined against the yeast, Saccharomyces cerevisiae. A clear structure-activity relationship was observed in these compounds. Thus, triarylbismuth dichlorides 2 [(4-YC6H4)3BiCl2: Y=MeO, F, Cl, CF3, CN, NO2] and halobismuthanes 6 [2-(t)BuSO2C6H4(4-YC6H4)BiX: Y=MeO, Me, H, Cl; X=Cl, Br, I], 7 [Bi(X)(C6H4-2-SO2C6H4-1'-): X=Cl, Br, I], 8 [2-Me2NCH2C6H4(Ph)BiX: X=Cl, Br] and 9 [4-MeC6H4(8-Me2NC10H6-1-)BiCl] showed the growth inhibition effect, while triarylbismuth difluorides 3 [(4-YC6H4)3BiF2] and triarylbismuthanes 1 [(4-YC6H4)3Bi], 4 [2-(t)BuSO2C6H4(4-YC6H4)2Bi] and 5 [4-YC6H4Bi(C6H4-2-SO2C6H4-1'-)] were not active at all irrespective of the nature of the substituents. Generation of the inhibition effect is governed by the facility of nucleophilic reaction at the bismuth center and the Lewis acidic bismuth center is an active site. Of all the bismuth compounds attempted, halobismuthanes 7 derived from diphenyl sulfone exhibited the highest activities. An X-ray crystallographic study of 7a [Bi(Cl)(C6H4-2-SO2C6H4-1'-)] revealed that the bismuth center adopts a seven-coordinated geometry, which is unusual in organobismuth(III) compounds, through the intramolecular and intermolecular coordination between the bismuth and oxygen atoms. The marked inhibition effect of 7 may be attributed to such a highly coordinated geometry, which allows the bismuth center to bind tightly with some biomolecules playing important roles in the growth of S. cerevisiae.     

Transition state analysis for human and Plasmodium falciparum purine nucleoside phosphorylases

Recent studies have shown that Plasmodium falciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1'-(3)H]-, [2'-(3)H]-, [1'-(14)C]-, [9-(15)N]-, and [5'-(3)H]inosines were 1.184 +/- 0.004, 1.031 +/- 0.004, 1.002 +/- 0.006, 1.029 +/- 0.006, and 1.062 +/- 0.002 for the human enzyme and 1.116 +/- 0.007, 1.036 +/- 0.003, 0.996 +/- 0.006, 1.019 +/- 0.005, and 1.064 +/- 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D(N)A(N) (S(N)1) stepwise mechanism with very little leaving group involvement. The near-unity 1'-(14)C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1'-(14)C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9-(15)N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-(15)N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small beta-secondary 2'-(3)H KIE for both enzymes indicates a 3'-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5'-(3)H KIE reveals substantial distortion at the 5'-hydroxymethyl group which causes loosening of the C5'-H5' bonds during the reaction coordinate.     

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-[(substituted azetidinone/thiazolidinone)-aminomethyl]-6-bromoquinazolin-4-ones as anti-inflammatory agent

N-Chloroacetyl-5-bromoanthranilic acid (1), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-chloromethyl-6-bromoquinazolin-4-one (2), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-hydrazinomethyl-6-bromoquinazolin-4-one (3), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-substitutedbenzylidene aminomethyl-6-bromoquinazolin-4-ones (4-11), 2-[(4'-oxo-3'-chloro-2'-phenylazetidin-1'-yl)aminomethyl]-3-[4'-(p-chlorophenyl)thiazol-2'-yl]-6-bromoquinazolin-4-ones (12-19) and 2-(4'-oxo-2'-phenyl-thiazolidin-3'-yl-aminomethyl)- 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-6-bromoquinazolin-4-ones (20-27) have been synthesized. All the compounds have been screened for their anti-inflammatory and analgesic activities at the dose of 50mg/kg po. Compound 21 showed maximum anti-inflammatory (38.35%) and analgesic (37.36%) activities. Compound 21 was also tested for ulcerogenic activity and the UD(50) value was found to be 195.6mg/kg po. The structure of all compounds has been evaluated by elemental analysis (C, H, N) and spectral analysis (IR, (1)H NMR and mass spectrometry).     

Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-K1 cells

The action of adriamycin (an inhibitor of precursor protein import into mitochondria) upon phosphatidylserine (PtdSer) import into mitochondria was examined in permeabilized CHO-K1 cells. The decarboxylation of nascent PtdSer to phosphatidylethanolamine was used as an indicator reaction for the lipid translocation process. Adriamycin was without effect upon new PtdSer synthesis but blocked the time- and translocation-dependent decarboxylation of this lipid at the mitochondrial inner membrane of permeabilized cells. The effect of adriamycin was concentration-dependent with an IC50 of 150 microM and was not due to direct inhibition of PtdSer decarboxylase. To determine at which level of PtdSer transport adriamycin was working, the adriamycin-treated permeabilized cells were incubated with 1-acyl-2-[N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl)] aminocaproyl)]phosphatidyl[1'-14C] serine (NBD-Ptd[1'-14C]Ser), and its decarboxylation was determined. Since the NBD-Ptd[1'-14C]Ser freely partitions into all cell membranes, it can partition into the outer mitochondrial membrane in an ATP-independent fashion. The NBD-Ptd[1'-14C]Ser was readily decarboxylated in an ATP-independent manner in permeabilized cells. Adriamycin inhibited the decarboxylation of NBD-Ptd[1'-14C]Ser, thereby indicating that it can act upon lipid transport processes between the outer and inner mitochondrial membrane.     

牛心朴子须根的化学成分研究

从采自宁夏的萝摩科鹅绒藤属植物牛心朴子 (CynanchumkomaroviiAl.Iljinski.)须根的乙醇提取物中分离鉴定了十个非C2 1 甾体类化合物 :β D 呋喃果糖基 (2→ 1) α D [6 O 芥子酰基 ] 吡喃葡萄糖甙 (1) ,β D (3 O 芥子酰基 ) 呋喃果糖基 (2→ 1) α D [6 O 芥子酰基 ] 吡喃葡萄糖甙 (2 ) ,[6 O β D 吡喃葡萄糖基 (1→ 6 ) β D 吡喃葡萄糖基 1,2 双氧 (4 羟基 3,5 二甲氧基肉桂酰 ) (3) ,7 脱甲氧基娃儿藤碱 (4) ,9 羟基 芳樟醇 3 O β D 吡喃木糖基 (1→ 6 ) β D 吡喃葡萄糖甙 (5 ) ,(2E ,6R) 2 ,6 二甲基 2 ,7 辛二烯 1,6 二醇 (6 ) ,[(+) 丁香素 ](7) ,4′ O demethylepiyangambin(8) ,4′ 羟基 2′ 甲氧基苯乙酮 (9) ,(2S ,3S ,4R ,12E) N [2′ (R) 羟基二十二碳烷基 ] 1,3,4 三羟基 2 酰胺 二十碳烷基 12 烯 (10 )。除化合物 4和 9外 ,其余化合物均为首次从该植物中分离得到。     

Stoichiometric measurement of the acrosin content of a guinea-pig spermatozoon

Guinea-pig spermatozoa were induced to undergo an acrosome reaction with the ionophore A23187. A time course for the activation of protease activity was established. Acid treatment of fully activated spermatozoa at pH 2.35 for 30 min exposed additional activity. This was attributed to the acid dissociation of a protein inhibitor from acrosin. The acrosin content of fully activated and acid-dissociated sperm extracts was measured using a sensitive active-site titrant for serine proteases. The number of acrosin molecules per spermatozoon, calculated on the basis of the sperm count, was approximately 2 x 10(6), of which half were available without dissociation of the inhibitor.