PubMed Health⌕ Search

Biomedical subjects

T C Liang

Publications and source records attributed to T C Liang.

12 recordsLinked to original sources
PubMed1996

Antibody binding to a peptide but not the whole protein by recognition of the C-terminal carboxy group.

Antipeptide antibodies have become indispensable tools in modern biochemistry and molecular biology. Unfortunately, not all antipeptide antibodies react with their target proteins. The reasons why certain antipeptide antibodies fail to do so are not always clear, although it is commonly assumed that conformational difference between the peptide antigens and the corresponding sequences in proteins accounts for most failures. Here, we report detailed characterization of an antipeptide mAb which reacted avidly with the peptide antigen but did not react with the same sequence in a protein. ELISA analysis using analogs of the antigen peptide revealed that this mAb did not react with a C-terminus-extended analog of the antigen peptide and reacted poorly with a peptide amide analog of the antigen peptide. These results suggest that the mAb recognizes an epitope including the C-terminal-free carboxyl group of the peptide. This analysis also revealed that the epitope recognized by this mAb was located in the C-terminal pentapeptide, RY-IRS. Four amino acid side chains (R,I,R, and S) in this pentapeptide were shown by alanine-scanning to be critical for antibody recognition. Analysis of the polyclonal antisera raised against this peptide revealed that antibodies reacting with this unique carboxyl-containing epitope are most abundant. This unexpected finding has since been shown in several other cases in this laboratory, suggesting that generation of antibodies that recognize carboxyl-containing artificial epitopes may be rather common. we also found that the use of a peptide amide (instead of peptide acid) antigen did not prevent a similar problem; in this case, the C-terminal amide became part of the epitope. Based on these findings, we suggest a method for enhancing the probability of isolating protein-reactive mAbs.

Amino Acid Sequence↗
PubMed1996

A universal tag for recombinant proteins.

Incorporation of tags into recombinant proteins can facilitate their identification and purification. In addition, these tags can also be used to monitor the trafficking or localization of the recombinant proteins inside the cells. Several such tags have been developed. However, the lengths of these tags make it cumber-some to incorporate them into the desired proteins. Typically, one must subclone the desired cDNA into a plasmid containing the tag sequence at a suitable restriction site or ligate a synthetic oligonucleotide containing the tag sequence at a suitable restriction site in the cDNA of the desired protein. These manipulations can be avoided, if one uses a short peptide tag that can be incorporated by PCR. We show here that a short peptide tag, RYIRS can be easily incorporated at the C termini of recombinant proteins by PCR. We also showed that by using a mAb specific for this peptide sequence, the tagged proteins could be easily detected in Western blot analysis, immunofluorescence staining, and immunoprecipitation. The interactions between this tag sequence and the mAb have been well characterized. One can take advantage of this information and control the reactivities between the tagged proteins and the mAb by varying the lengths of the peptide tags. Furthermore, we showed that this tag can be used to monitor whether a recombinant protein is properly translated and terminated because the interactions between this tag sequence and the mAb requires that the tag be at the C-terminus of the protein.

Amino Acid Sequence↗
PubMed1994

Substrate specificity of porcine renin: P1', P1, and P3 residues of renin substrates are crucial for activity.

Renin, the rate-limiting enzyme in the formation of angiotensin II, is well-known for its stringent substrate specificity. In this study, the biochemical basis for the unusual specificity of renin was investigated by replacing individual amino acids in the octapeptide substrate of renin with Ala. Kinetic analyses of Ala-substituted substrates revealed that the substitutions did not cause significant changes in the Km values, but did cause variable changes in the kcat and kcat/Km values. Ala substitutions at the P1', P1, and P3 sites decreased the kcat/Km values by 400-700-fold. Similar substitutions at the P3', P2, P4, and P5 sites only reduced the kcat/Km values by 2-7-fold. Interestingly, Ala substitution for the P2' Val produced a substrate with an approximately 3-fold increase in activity. These results indicate that the P1', P1, and P3 residues are crucial in determining the substrate specificity of renin. The findings also suggest that the specificity of renin is achieved mainly through substrate discrimination in the transition state, rather than in the ground state. Further studies on the effects of amino acid substitutions at the P2' site revealed that non-branched-chain amino acids (e.g., Ala and alpha-aminobutyric acid) are preferred at this site. Only P1' substitution demonstrated any significant change in Km, presumably due to the decreased hydrophobic interactions in the S1' site upon Ala substitution. The species specificity of renin presumably arises from differing P1'-P3' residues in angiotensinogens. For example, the P1'-P3' residues from human and porcine angiotensinogens are Ile-Val-His and Leu-Val-Tyr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1994

Three-dimensional structure of chymotrypsin inactivated with (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane: implications for the mechanism of inactivation of serine proteases by chloroketones.

The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Fluorogenic peptides containing only alpha-amino acids.

Fluorogenic substrates for endopeptidases are often prepared by attaching fluorophores and quenchers to opposite ends of the peptide substrates. Here, we describe a new approach by incorporating tryptophan and p-nitrophenylalanine into peptides to give fluorogenic substrates composed entirely of alpha-amino acids. Two advantages are apparent: (1) They can be prepared on a peptide synthesizer; no synthetic expertise is required. (2) Both ends of these peptides are free; other residues can be attached to increase their solubilities or to label them with affinity ligands. We tested the applicability of this new approach by developing a continuous assay for renin, a protease with unusually stringent substrate specificity. The fact that new fluorogenic peptides exhibited similar kinetic properties as those of known renin substrates suggests that this new method should be generally applicable to other proteases.

Amino Acid Sequence↗
PubMed1994

Studies on the specificity of acetylaminoacyl-peptide hydrolase.

In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1993

Conformation dependence of antipeptide antibodies: characterization of cell-CAM105 isoform-specific antipeptide antibodies using proteins expressed in insect cells with baculoviral vectors.

Cell-CAM105 proteins are hepatocyte adhesion molecules of the immunoglobulin superfamily. The two isoforms, L-form and S-form, are highly homologous. In their extracellular domains, only 16 amino acid substitutions are found scattered in the first immunoglobulin domain of 105 amino acids. Peptide sequences containing these differences are selected for production of antibodies. Isoform specificities of these antibodies were evaluated with proteins expressed in the baculovirus-insect cell system. In immunoblot and immunoprecipitation experiments, anti-C1 antibody, which was generated against a peptide sequence found only in the cytoplasmic domain of the L-form, reacted only with the L-form cell-CAM105 protein. Anti-N antibody, which was generated against a pentadecapeptide of the L-form, reacted with both the S- and L-isoforms. The lack of isoform specificity of anti-N is not surprising because there is only one amino acid substitution in this pentadecapeptide sequence. In contrast, S3, a S-form-specific pentadecapeptide with four amino acid substitutions was able to elicit antibody, anti-S3, specific for the S-isoform. With the commonly used immunoprecipitation procedures, only anti-C1 was able to precipitate cell-CAM105 from the liver membrane. Anti-N and anti-S3 could precipitate the proteins only after the liver membrane samples had been boiled in the presence of denaturing agents. Hydropathy analysis of these peptides revealed that both peptides N and S3 are more hydrophobic than peptide C1, suggesting that the peptide fragments N and S3 are probably not located on the surface of the protein. This may explain why boiling of the protein sample was necessary before anti-N and anti-S3 could precipitate the protein. The present study demonstrates that it is possible to produce isoform-specific antibodies for highly homologous proteins. Furthermore, we show that special sample treatment may be required to expose the antigenic sites.

Adenosine Triphosphatases↗
PubMed1989

pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone.

The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK's at 7.0 and 8.9. We assign these pK's to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang & Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK greater than 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK less than 4.9). The pK of His 57 is increased by greater than 3 units over that in the free enzyme, and the pK of the hemiketal decreased by greater than 4 units compared to the pK in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1987

Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy.

A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.

Carbon Isotopes↗
PubMed1987

Inhibition of papain by nitriles: mechanistic studies using NMR and kinetic measurements.

N-(N-acetyl-1-phenylalanyl)aminoacetronitrile is an inhibitor of papain. With 13C NMR spectroscopy we have shown that a reversible covalent adduct is formed with papain. The reversible nature of the covalent-adduct formation was demonstrated with NMR saturation-transfer technique using a DANTE pulse for selective excitation. In addition the covalent adduct was displaced with an aldehyde inhibitor to regenerate the nitrile compound. No hydrolysis of the nitrile was observed. The covalent adduct is most likely a thioimidate formed between the essential thiol and the nitrile. Several p-nitroanilide substrates and their corresponding nitrile inhibitors were examined. A correlation between Ki and kcat/Km was observed. This finding together with the fact that the pH dependence of Ki parallels that of kcat/Km suggests that the interaction of nitriles and papain has considerable transition-state character. In contrast, a nitrile was shown to be an ineffective inhibitor of alpha-chymotrypsin.

Acetamides↗
PubMed1975

Effects of pentanoic acid and 4-pentenoic acid on the intracellular fluxes of acetyl coenzyme A in Tetrahymena.

Cultures of Tetrahymena pyriformis were incubated for 1 hour with a mixture of acetate, pyruvate, and pentanoate with only one substrate labeled at a time and with the position of the label chosen so that [1-14-C]acetyl coenzyme A was an early product of the metabolism of each substrate. The appearance of label in CO2, lipids, glycogen, glutamate, and alanine were measured and results interpreted in terms of a previously developed three-compartment model of metabolism, which was found to quantitatively describe the data even when two of the flux rates (the flux of acetyl-CoA from the peroxisomal to the outer mitochondrial compartment and from the outer mitrochondrial to the inner mitochondrial compartment) were set equal to zero. This reduction in the number of independent parameters leads to the model being overdetermined and to a probably unique fit of the three-compartment model tof the present data and to previous data when octanoate was the fatty acid substrate. Pentanoate was metabolized to a greater extent than octanoate and did not inhibit growth. Pentanoate inhibited acetate utilization in both the inner mitochondrial and peroxisomal compartments as indicated by a reduction in the incorporation of label from [1-14-C]acetate into lipids and into CO2, but there was no difference in oxidation of [2-14-C]pyruvate when pentanoate was the fatty acid substrate as compared to octanoate. Glyconeogenesis was inhibited when pentanoate was substituted for octanoate. Similar experiments were performed on cells treated with 4-pentenoic acid. The effects of 4-pentenoic acid were essentially the same whether octanoate or pentanoate was the fatty acid substrate, i.e. inhibition of glyconeogenesis from all labeled substrates and inhibition of [2-14-C]pyruvate oxidation. The results indicate that the effects of pentanoate are largely confined to the peroxisomal and the inner mitochondrial compartments whereas the effects of 4-pentenoic acid are confined to the peroxisomal and outer mitochondrial compartments.

Acetates↗