Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Thiol-blocking reagents and phosphate acetyltransferase catalysis, and the assessment of protection by adsorbed molecules (Short Communication)

Phosphate acetyltransferase (EC 2.3.1.8) is shown to be unusually sensitive to thiol-blocking reagents, and one or more thiol groups probably catalyse the surface reaction. A novel method for assessing protection by adsorbed species is described. The adsorbed substrate molecules acetyl-CoA and phosphate are found to provide substantial protection against thiol-blocking reagents.     

Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.     

The sulfhydryl groups responsible for bilitranslocase transport activity respond to the interaction of the carrier with bilirubin and functional analogues

Both inactivation of sulfobromophthalein transport in rat liver plasma membrane vesicles by sulfhydryl group reagents and subsequent reactivation by 2-mercaptoethanol are shown to be modulated by ligands to bilitranslocase. In particular, bilirubin, sulfobromophthalein and Thymol blue behave as negative effectors in the inactivation reaction and as positive effectors in the reactivation reaction. Kinetic data provide further evidence of the existence of two classes of sulfhydryl groups involved in transport activity. The effect brought about by remarkably low concentrations of bilirubin is in line with the physiological function of bilitranslocase as a bilirubin carrier.     

Modification of tRNA 1 Val from yeast with monoperphthalic acid]

A method is proposed for analysis of natural and chemically modified polynucleotides which consists in enzymatic conversion of the polymer or oligomer into nucleosides followed by cation-exchange chromotography on the microcolumns. By using the method developed it was shown that after treatment of the yeast tRNAVal and tRNAPhe with monoperphthalic acid N-oxides of adenosine and cytidine were formed. Poly (U, G) was not modified at a measurable extent whereas GMP was decomposed. In tRNAVal (yeast)the adenosines and cytosines of the anticodon loop and 3'-end are most reactive; it is the case for the C17 of the diHU-loop as well. These data are in agreement with the results obtained for tRNA modification with other reagents and for limited enzymatic hydrolysis of the tRNAVal. The limitations of the reaction of the monoperphthalate with nucleic acids are briefly discussed.     

Capping revisited: II. Low sensitivity to sulfydryl reagents

While thiols had been shown to either delay capping of lymphocyte membrane components or to leave it unaffected, sulfydryl reagents can block the process. However, capping is not completely blocked by doses of SH reagents below 10^?3M and thus, appears to be particularly resistant to SH poisoning. The extent of inhibition is concentration dependent, and in the lower dose range, some facilitation of capping can even be obtained. The effects of the drugs on the capping process actually depend on their reaction with SH radicals as shown by competition with free thiols. Some of the SH reagents used here are known inhibitors of lymphocyte triggering. There is no direct correlation, however, namely, there is in general rather little inhibition of capping at doses of SH reagents sufficient to completely abolish triggering.     

Effect of mass-transfer limitations on the selectivity of immobilized alpha-chymotrypsin biocatalysts prepared for use in organic medium

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Shelf Life of Fluorescent Treponemal Antibody-Absorption Test Reagents

Properly prepared, standarized, and stored fluorescent treponemal antibody-absorption (FTA-ABS) reagents have been shown to have stabilities equal to other biological reagents. A liquid antigen over 10 years old has been shown to give a satisfactory reaction. Newer preparations have now been shown to be stable for over 5 years, and the tests on each are being continued. The very new liquid antigens which were originally standardized by the FTA-ABS method have shown no decrease in potency over a 20-month period. Stability studies on antigens dried on slides are now in their eighth month, with no apparent loss in potency. The stability of the conjugate is constant when stored frozen at -20 C or lyophilized. When stored as a liquid at 4 C, the stability is governed by the pH and the molarity of the buffer. The standardized and lyophilized sorbent has been shown to be stable for over 1 year.     

Photoaffinity reagents for use with pepsin and other carboxyl proteases

Two compounds have been designed to serve as photoaffinity reagents for use with carboxyl proteases. 1,2-Epoxy-3-(4'-azido-2'-nitrophenoxy)propane has been synthesized and shown to react with porcine pepsin in the same fashion as the traditional inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane, while p-azidophenacyl bromide is similar to other phenacyl bromides in its reaction with pepsin. In combination with p-azido-alpha-diazoacetophenone, previously shown to resemble alpha-diazo carbonyl reagents in its reaction with pepsin, photoaffinity analogs are now available for all three of the widely-used carboxyl protease inhibitors.     

Topological information content and chemical reactions

The difference of the topological information content of two reacting molecules and that of their reaction products is calculated for several topological types of chemical reactions, illustrating the influence of the structure of the reagents and of the reaction product. It is shown that the change in the topological information content in a chemical reaction can be positive as well as negative, depending on the way the reagents approach each other and thus on the reaction product formed. A quantitative measure of structural specificity is introduced.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Pregnancy initiation in the rhesus macaque: Towards functional manipulation of the maternal-fetal interface

Nonhuman primates provide an important opportunity to define the mechanisms that contribute to the success of early pregnancy. We have focused for several years now on defining the expression of novel placental major histocompatibility complex (MHC) class I molecules. In parallel, we have used reagents against human immune cell markers to characterize the leukocyte population in the decidua and have demonstrated dynamic changes in these cell populations during the first 5 weeks of gestation. The challenge is to identify the possible role(s) of placental MHC class I in modifying/directing the maternal endometrial or systemic immune system in the post-implantation period. Foremost among the challenges is the difficulty in modifying placental function. In the instance of trophoblast surface proteins, passive immunization studies are feasible, although limitations include the empirical nature of this approach, as well as the inability to modify intracellular function. We have shown that using lentiviral vectors to effect preimplantation gene transfer for transgene expression in the placenta is not only feasible, but of good efficiency. In addition to transgene overexpression, robust approaches for knocking down/knocking out placental gene expression are essential. Recent developments in RNA interference approaches may allow "transient knockout" experiments. While the rhesus monkey has been our model of choice, currently there are limitations in the number of available female rhesus monkeys of reproductive age for research in early pregnancy. It is critical that the technologies for advanced study move forward in other species. The baboon has been used significantly in reproductive tract biology and early pregnancy research and important models have been developed for manipulation of the maternal-fetal interface. Additional characterization of other species, such as the cynomolgus and African green (vervet) monkey is critical. Given the limitations on antigen recognition when using human reagents, we also propose that the development of panels of primate-specific anti-leukocyte antibodies is essential for moving forward nonhuman primate reproductive research.     

Kinetics of the aspartate-aminotransferase reaction catalyzed by free and immobilized cells of E. coli]

The kinetics of the aspartate-aminotransferase reaction were studied, using free and immobilized cells of E. coli, strain 85 as an enzyme source. It was shown that the reaction is limited by mass transport of the reagents through the bacterial cell membrane even at high concentrations of the substrates in the surrounding solution. The polyacrylamide gel-incorporated cells of E. coli, strain 85 catalyze the aspartate-aminotransferase reaction more effectively as compared to free or destroyed cells. In the latter case the reaction is characterized by the following kinetic parameters: the effective values of the stationary rate of the product accumulation and its stationary efflux from the cell are equal to (15,37 +/- 0.4) . 10(-6) mole/s/mg of protein and (3,01 +/- 0,8) . 10(-20) mole/s per 1 cell. respectively. The steady-state constant for glutamate synthesis from aspartic acid is equal to 0,22--0,23.     

The intrinsic pKa-values of functional groups in enzymes: improper deductions from the pH-dependence of steady-state parameters.

The assumptions implicit in the deductions made from the pH-dependence of rate measurements of enzyme-catalyzed reactions are summarized, and the limitations of such determinations are discussed nonalgebraically. The following types of pH-profile are considered (in order of increasing utility): pH-"activity" curves at fixed [S] o;pH-dependences of kcat, Km, kcat/Km, and Ki; pH-dependences of kmodification (by specific reagents) and of competitive labeling (by nonspecific reagents); pH-dependences of elementary steps; and the direct observation of a titrating group.     

The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter

The binding affinity of the cocaine analog [(3)H]2 beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT.     

The sulfhydryls of firefly luciferase are not essential for activity

Firefly luciferase, containing an average of seven free sulfhydryls per two 50 000-dalton polypeptides, was modified by various sulfhydryl reagents. The differential reactivities of the sulfhydryls in luciferase protected by substrates allow one to define three categories of these groups: Class SH-III contains three sulfhydryls that are not involved in enzymatic activity. Class SH-II contains two sulfhydryls whose modification by different reagents causes varying effects on activity ranging from 0 to 60% inactivation. These sulfhydryls are not essential but may be important structurally or sterically. Class SH-I contains two sulfhydryls that are protected by substrates, either dehydroluciferyl adenylate or dehydroluciferin alone, and are located at or near the active site. The SH-I sulfhydryls are vicinal in the enzyme as demonstrated by their ability to form a disulfide bond. They have also been shown to exist on a single polypeptide chain. Modification of the SH-I groups by most reagents results in complete loss of enzymatic activity; reaction with methyl methanethiosulfonate produces an enzyme that emits only red light whereas native luciferase emits yellow-green light. Evidence is presented that the modified enzyme, while catalytically active, has a distorted active site. It is concluded that these two SH-I sulfhydryls are not essential for activity.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Effects of different fuchsin analogs on the Feulgen reaction

The Feulgen reaction is used for cytophotometric quantitation of nuclear DNA. Schiff's reagents used in the Feulgen reaction usually are prepared from basic fuchsin, a variable mixture of four triaminotriphenylmethane analogs. The effect of the several fuchsin analogs on the quality of Schiff's staining of hydrolyzed DNA is not known. In this investigation Schiff's reagents prepared from relatively pure fuchsin analogs were used to determine whether different fuchsin analogs affect the absorbance of the Schiff's reagent-DNA complexes formed in solution. It has been determined that the complex formed by pararosaniline-Schiff's reagent and hydrolyzed DNA exhibits lower absorption than do corresponding complexes formed by Schiff's reagents prepared from magenta II or from new fuchsin.     

Reaction of the indole group with malondialdehyde: application for the derivatization of tryptophan residues in peptides

A method for the selective modification of tryptophan residues based on the reaction of malondialdehyde with the indole nitrogen of the tryptophan side chain at acidic conditions is presented. The condensation reaction is quantitative and leads to a substituted acrolein moiety with a remaining reactive aldehyde group. As is shown, this group can be further converted to a hydrazone using hydrazide compounds, but if hydrazine or phenylhydrazine are used, release of the free indole group is observed upon cleavage of the substitution. Alternatively, secondary amines such as pyrrolidine may also act as cleavage reagents. This general reaction scheme has been adapted and optimized for the derivatization of tryptophan-containing peptides and small N-heterocyclic compounds. It serves as the basis of a reversible tagging scheme for Trp-peptides or molecules of interest carrying indole structures as it allows the specific attachment and removal of a reactive group that may be used for a variety of purposes such as affinity tagging.