Kinetics and free energy profiles of spermine transport in liver mitochondria.

In the present study, the voltage-dependent mechanism of spermine transport in liver mitochondria [Toninello, A., Dalla Via, L., Siliprandi, D., and Garlid, K. D. (1992) J. Biol. Chem. 267, 18393-18397] was further characterized by determining the rate constants J(max) and K(m) as functions of membrane potential. An increase in mitochondrial membrane potential from 150 to 210 mV promoted spermine transport, as reflected by an approximate 4-fold increase in J(max) and 25% decrease in K(m). The mechanism for the voltage dependence of transport was examined using the beta value, i. e., the slope of ln(flux) vs FDeltaPsi/RT plots. Flux-voltage analyses performed at very high and very low spermine concentrations yielded beta values of 0.125 and 0.25, for J(max) and J(max)/K(m), respectively. The physical significance of these beta values was analyzed by means of a theory relating the enzyme reaction rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 303, 305-311]. Depending on the nature of K(m), two possible models could be proposed to describe the location and shape of the barriers in the membrane. Analysis of previous data concerning spermine binding [Dalla Via, L., Di Noto, V., Siliprandi, D., and Toninello, A. (1996) Biochim. Biophys. Acta 1284, 247-252] by a new rationale provided evidence for an asymmetrical energy profile composed of two peaks with the binding site near the membrane surface followed by a rate-determining energy barrier for the movement of the bound spermine toward the internal region of the membrane.

Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model.

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.

Histamine monoclonal antibody for brain immunocytochemistry.

Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AHA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara et al. (1997) Histochem. Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable cross-reaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki et al. [(1988) Brain Res. 439, 402-405; (1990) Exp. Brain Res. 80, 374-380] and Panula et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988) J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.

Enzyme kinetics based on free-energy profiles.

A comprehensive theory for relating free-energy profiles to kinetic equations of enzyme reactions has been developed. It enables expression of the overall rate and the concentrations of reaction intermediates in terms of the heights of peaks in free-energy profiles for various reactions. The reactions include consecutive reactions with intermittent irreversible steps, those with dead-end side reactions and completely reversible reactions. The usefulness of the theory is shown by analysis of a single-substrate reaction, a reaction with a covalent intermediate, and product inhibition in a two-substrate reaction, in which kinetic parameters such as Vmax and Km are related to the peak heights in free-energy profiles. The paper refers also to the concept of rate-determining step (RDS) and shows the utility of a related concept rate-determining zone (RDZ) which indicates the reaction steps between the main intermediate and the RDS. The concept of RDZ is useful for resolving confusion related to the concept of RDS. It should be emphasized that the present approach is applicable only to linear reactions.

Studies on enzymic condensation of long chain peptides.

Our recent researches toward enzymic condensation of long chain peptides are discussed. These involve two methods of peptide C-terminal activation and transpeptidation catalyzed by clostripain, trypsin and their derivatives.

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity.

Clostripain catalyses the transpeptidation between benzoylarginin ethyl ester and amino acid amides, oligopeptides, insulin A- and B-chains and tryptic peptides of myoglobin at millimolar substrate concentrations. The reactions proceed with temporary accumulation of the products, followed by hydrolytic decomposition. The yield was not affected significantly by the type of N-terminal amino acid, but was diminished markedly by the negative charges of the amine components. The yields for natural peptides were linearly related to the charge density of the peptides.

Two types of rate-determining step in chemical and biochemical processes.

Close examination of the concept of the rate-determining step (RDS) shows that there are two types of RDS depending on the definition of 'rate'. One is represented by the highest peak of the free-energy diagram of consecutive reactions and holds true where the rate is defined in terms of the concentration of the first reactant. The other is represented by the peak showing the maximum free-energy difference, where the free-energy difference is the height of a peak measured from the bottom of any preceding troughs, where the definition of the rate is in terms of the total reactant concentration including intermediates. There are no criteria a priori for selecting one of them.

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using beta-D-galactosidase coupled to a monoclonal antibody of IgM isotype.

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor.

A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.

Studies on protein semisynthesis. I. Formation of esters, hydrazides, and substituted hydrazides of peptides by the reverse reaction of trypsin.

An enzymatic method for converting tryptic peptides into their intermediates for the azide method of peptide synthesis was investigated by using model substrates, Bz-Arg, Bz-Gly-Arg, and Bz-Gly-Lys. The conditions favorite for the formation of the esters, hydrazides, and substituted hydrazides were deduced from the theoretical analysis on the pH and pKa dependence of the synthetic reactions and the pKa values of the substrates in various solvents. The theory based on the empirical relationship between the pKa of amines and the formation constant of the amides (Fersht, A.R. & Requena, Y. (1971) J. Am. Chem. Soc. 93, 3499-3504) indicates that the most favorite amine in the formation of amides is the one with a pKa value equal to the pKa of the carboxyl group and with such amines the amide formation takes place almost quantitatively even in aqueous solutions. Boc- and Cbz-hydrazine were found to satisfy the above condition approximately. The methyl esters of the substrates were formed in 30-35% yields in 50% aqueous methanol at pH near 4.5 by the reverse reaction of trypsin [EC 3.4.4.4]. Bz-Gly-Arg-NHNH2 was formed in a 34% yield in 2 M aqueous hydrazine and in 70-80% yields in 50% aqueous solutions of dioxane, DMF, and DMSO containing 1 M hydrazine at pH near 7. Bz-Gly-Arg-NHNH-Boc and Bz-Gly-Lys-NHNH-Boc were formed quite readily in 90-98% yields in H2O and in 50% aqueous solutions of dioxane, DMF, and DMSO containing 0.5 or 1 M Boc hydrazine at pH from 4 to 5. The Cbz-hydrazides of the peptides were also formed readily in 88-95% yields in the above mixed solvents containing 0.5 M Cbz-hydrazine. The formation constants of the hydrazides in H2O were in good agreement with those predicted by the empirical relationship. The formation constants in the form of log Ke in the mixed solvents calculated by taking the activity coefficients of the reactants as unity were 0.1 to 0.5 lower than the predicted values.

Mercuri-nitrophenol as a reporter group for the conformational change of hemoglobin.

One mole of horse hemoglobin tetramer reacts with 2 moles of 2-chloromercuri-4-nitrophenol (MNP) at beta 93 cysteine. The difference spectra between NMP-bound hemoglobin and hemoglobin, measured with the aid of ascorbic acid and ascorate oxidase [EC 1.10.3.3] as deoxygenation reagents, indicate that the pK of the phenolic hydroxyl group of MNP increases by 0.6 to 0.8 pH unit on deoxygenation of the hemoglobin. The Hill constant of the modified hemoglobin changes with pH. It decreases from about 2.4 at pH 6.8 to about 1.0 at pH 9.0 This effect of the reagent is interpreted as inherent to the reporter groups.

A method for observing protein-protein interaction.

A method is proposed for observation of the interaction between charged macromolecules such as proteins. The method is based on the fact that the pK of an ionizable reporter group attached to a macromolecule can be altered by the electrostatic effect of another charged macromolecule which associates with the former. The effectiveness of the method was shown in the study of the association of bovine serum albumin with hen egg lysozyme [EC 3.2.1.17]. The errors inherent in this method in obtaining the equilibrium constant of the association reaction and procedures for their correction are discussed.