Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure

Reaction of protein amino groups with glucose (the Maillard reaction) leads from early stage products such as Schiff base and Amadori products to advanced glycation end products (AGE), structures implicated in diabetic complications and the aging process. We have prepared the polyclonal anti-AGE antibody and the monoclonal anti-AGE antibody against AGE-bovine serum albumin and made an immunochemical approach to characterize AGE structures. Both polyclonal and monoclonal antibodies reacted with AGE-proteins such as AGE-bovine serum albumin, AGE-human serum albumin, and AGE-hemoglobin but not with unmodified counterparts. Treatments of these AGE-proteins with borohydride had no effect on the immunoreactivity. Moreover, fructosyl-epsilon-caproic acid, a synthetic Amadori compound, did not serve as an antigen, indicating that these antibodies were specific for AGE products but not for early stage products of the Maillard reaction. In addition, these antibodies were also able to recognize AGE products prepared either from alpha-tosyl-1-lysine, alpha-tosyl-1-lysine methyl ester, monoaminocarboxylic acid such as epsilon-aminocaproic acid, gamma-amino-n-butyric acid, and beta-alanine. Thus, these results strongly suggest the presence of a common structure in AGE preparations, regardless of whether AGE products are generated from proteins, amino acids, or monoaminocarboxylic acids.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

Polymorphisms distinguishing different mouse species and t haplotypes.

Three anonymous chromosome 17 DNA markers, D17Tu36, D17Tu43, and D17Le66B, differentiate between house mouse species and/or between t chromosomes. The D17Tu36 probe, which maps near the Fu locus and to the In(17)4 on t chromosomes, identifies at least 15 haplotypes, each haplotype characterized by a particular combination of DNA fragments obtained after digestion with the Taq I restriction endonuclease. Ten of these haplotypes occur in Mus domesticus, while the remaining five occur in M. musculus. In each of these two species, one haplotype is borne by t chromosomes while the other haplotypes are present on non-t chromosomes. The D17Tu43 probe, which maps near the D17Leh122 locus and to the In(17)3 on t chromosomes, also identifies at least 15 haplotypes in Taq I DNA digests, of which nine occur in M. domesticus and six in M. musculus. One of the nine M. domesticus haplotypes is borne by t chromosomes, the other haplotypes are borne by non-t chromosomes; two of the six M. musculus haplotypes are borne by t chromosomes and the remaining four by non-t chromosomes. Some of the D17Tu43 haplotypes are widely distributed in a given species, while others appear to be population-specific. Exceptions to species-specificity are found only in a few mice captured near the M. domesticus-M. musculus hybrid zone or in t chromosomes that appear to be of hybrid origin. The D17Leh66B probe, which maps to the In(17)2, distinguishes three haplotypes of M. domesticus-derived t chromosomes and one haplotype of M. musculus-derived t chromosomes. Because of these characteristics, the three markers are well suited for the study of mouse population genetics in general and of t chromosome population genetics in particular. A preliminary survey of wild M. domesticus and M. musculus populations has not uncovered any evidence of widespread introgression of genes from one species to the other; possible minor introgressions were found only in the vicinity of the hybrid zone. Typing of inbred strains has revealed the contribution of only M. domesticus DNA to the chromosome 17 of the laboratory mouse.     

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.     

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.     

The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.     

Projection of Monte Carlo and molecular dynamics trajectories onto the normal mode axes: human lysozyme

A method is presented to describe the internal motions of proteins obtained from molecular dynamics or Monte Carlo simulations as motions of normal mode variables. This method calculates normal mode variables by projecting trajectories of these simulations onto the axes of normal modes and expresses the trajectories as a linear combination of normal mode variables. This method is applied to the result of the molecular dynamics and the Monte Carlo simulations of human lysozyme. The motion of the lowest frequency mode extracted from the simulations represents the hinge bending motion very faithfully. Analysis of the obtained motions of the normal mode variables provides an explanation of the anharmonic aspects of protein dynamics as due first to the anharmonicity of the actual potential energy surface near a minimum and second to trans-minimum conformational changes.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.