Inhibition of passive cutaneous anaphylaxis by synthetic human immunoglobulin E peptide fragments.

In an attempt to determine the regions responsible for type I immediate hypersensitivity, a total of 42 peptide fragments, which cover the CH3-CH4 domains in human immunoglobulin E (IgE), were chemically synthesized. Several peptide fragments located in the amino acid sequences Ala329-Thr357 and Arg419-Ala463, inhibited passive cutaneous anaphylaxis (PCA) in vivo. In order to pinpoint the sites responsible for the inhibition of the PCA reaction, various fragment peptides in these two regions were synthesized. As a result, residues Pro343-Leu348, Pro426-Thr433, and Ser456-Thr461 were suggested to be involved in type I immediate hypersensitivity.

Inhibition of passive sensitization of human peripheral basophils by synthetic human immunoglobulin E peptide fragments.

To delineate the binding site in the human immunoglobulin E (IgE) molecule to the Fc epsilon receptor on basophils and mast cells, we chemically synthesized a total of 71 peptide fragments within the sequence Ser300-Lys547 in the human IgE molecule. The synthetic peptides were tested for their capacity to inhibit passive sensitization of human peripheral basophils with atopic patient's serum containing the specific IgE against dust mites in vitro. It was found that a peptide fragment, Pro345-Ile356, potently inhibited the passive sensitization. To clarify the minimal active core, various analogues, such as shortened, substituted (by Gly or Ala residue), omission and retro-sequence peptides, were synthesized and assayed. The results suggested that the sequence Pro345-Lys352 in the human IgE molecule would be an IgE binding site, and that a synthetic octapeptide, Pro345-Phe-Asp-Leu-Phe-Ile-Arg-Lys352, inhibited the passive sensitization, probably by occupying the Fc epsilon receptor sites on the cells.

Cross-linking of contractile proteins from skeletal muscle by treatment with microbial transglutaminase.

The action on muscle proteins of microbial transglutaminase (MTGase), which catalyzes the formation of a "zero-length" covalent cross-link between glutamine and lysine residues in peptides, was studied in order to define a basis for future application of MTGase cross-linking to the study of muscle protein interaction. We examined the cross-linking of skeletal muscle myosin, myosin subfragments, actin, and myofibrils by treatment with MTGase and the possible side-effects of the cross-linking on the enzymic activity of myosin, and found that the rod portions of myosin in myosin filaments were quickly cross-linked to each other by the action of MTGase, but myosin subfragment 1 was not cross-linked to actin. The MgATPase activities at 0.5 M KCl of myosin, heavy meromyosin, subfragment 1, and subfragment 1-actin were not significantly affected by the MTGase reaction. A very small fraction of the head portion of heavy meromyosin was cross-linked to actin in their rigor complexes by MTGase, and the ATPase activity at 0.5 M KCl of the cross-linked heavy meromyosin-actin complexes was slightly enhanced.

Effects of enterostatin (Val-Pro-Asp-Pro-Arg) on fat intake and blood levels of glucose and insulin in rats.

Enterostatin may be involved in the preference for fat and the control of fat intake. Using two different feeding patterns, we observed a change in food intake after injection of enterostatin (VPDPR) into the third ventricle. When rats were adapted to free selection choice between low fat (LF) and high fat (HF) diets, VPDPR inhibited intake of the LF diet at 100, 200 and 800 ng and inhibited intake of the HF diet at 200 ng. The dose-response of HF diet intake to VPDPR was U-shaped. However, even the optimal dose (200 ng), which reduced the intake of both LF and HF diets when both diets were given together, was not effective when the LF diet was given alone. In the present study, VPDPR has also shown to not affect plasma glucose or insulin levels. These results suggest that exogenous VPDPR may inhibit appetite when endogenous enterostatin secretion is increased by ingestion of dietary fat, and that VPDPR has a limited range of effects on feeding behavior. We support the hypothesis that the early satiety sense of VPDPR as an anorectic agent in a central site is directly related to endogenous enterostatin or procolipase levels after fat intake, but not glucose or insulin levels.

Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form.

(MTG) The Streptoverticillium transglutaminase gene, synthesized previously for yeast expression, was modified and resynthesized for overexpression in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200-300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.

Purification and calcium dependence of transglutaminases from sheep hair follicles.

To study the calcium sensitivity of sheep hair follicle transglutaminase, which was reportedly calcium-independent [H. W. Harding and G.E. Rogers, Biochemistry, 11, 2858-2863 (1972)], the enzyme was purified from a homogenate of merino sheep hair follicles and its calcium dependence was examined. As a result of purification, two types of transglutaminases (DEAE-unabsorbed and absorbed transglutaminase, DU-TG and DA-TG, respectively) were obtained. The molecular mass of DU-TG was 77 and 82 kDa by SDS-PAGE and gel filtration, respectively, while that of DA-TG was 40 and 80 kDa. Each enzyme was obviously calcium dependent and contained (a) cysteine residue(s) in the active site, like other known mammalian transglutaminases. Maximum activation of DU-TG and DA-TG was observed at 1 and 0.1 mM CaCl2, respectively.

Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.

Effect of chemical modifications on freeze denaturation of lactate dehydrogenase.

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.

The epsilon-(gamma-glutamyl)lysine moiety in crosslinked casein is an available source of lysine for rats.

To determine bioavailability, expressed as the protein efficiency ratio (PER) and biological value (BV) in rats, of the epsilon-(gamma-glutamyl)lysine [epsilon-(gamma-Glu)Lys] moiety in crosslinked proteins, we prepared heavily crosslinked [21.5 /micromol epsilon-(gamma-Glu)Lys/g casein] and intermediately crosslinked [13.6 micromol epsilon-(gamma-Glu)Lys/g casein] casein, using microbial transglutaminase. In Experiment 1, rats were assigned to one of four diets (heavily or intermediately crosslinked caseins, intact casein or non-protein diet) for 4 wk to evaluate the bioavailability of the epsilon-(gamma-Glu)Lys moiety in crosslinked casein as the sole source of dietary protein. Rats that were fed intact casein and the two crosslinked caseins had similar growth rates, PER, and BV, indicating that crosslinked caseins supported the growth of rats similarly to the intact casein. In Experiment 2, heavily crosslinked casein was added to wheat gluten-based diets in concentrations of 20 and 40 g/kg diet to evaluate the bioavailability of lysine in the epsilon-(gamma-Glu)Lys moiety of the casein as a lysine supplement for lysine-poor gluten. One of six diets (heavily crosslinked or intact casein diets in the two concentrations, gluten diet, or non-protein diet) was fed to rats for 4 wk. No significant differences in food intake, body weight gain, PER or BV were observed among rats fed the intact or crosslinked casein diets at either 2 or 4 g/100 g casein. These results suggest that the epsilon-(gamma-Glu)Lys moiety in crosslinked caseins are absorbed and therefore supplement the gluten. HPLC analysis of urine and feces of rats fed the crosslinked caseins actually confirmed that approximately 99% of the epsilon-(gamma-Glu)Lys moiety was absorbed in the body.

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Transglutaminase-induced cross-linking between subdomain 2 of G-actin and the 636-642 lysine-rich loop of myosin subfragment 1.

G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The cross-linking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636-642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038-14046). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2 mM MgCl2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATPase activity of the cross-linked S1 was not activated by actin, whereas the K+ (EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.