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B Haribabu

Publications and source records attributed to B Haribabu.

35 records · Page 2Linked to original sources
PubMed1996

Cross-desensitization among receptors for platelet activating factor and peptide chemoattractants. Evidence for independent regulatory pathways.

Cross-desensitization among receptors for peptide chemoattractants have been shown to involve two independent processes, receptor phosphorylation and inhibition of phospholipase C (PLC) activation. Receptors for lipid chemoattractants, i.e. platelet activating factor (PAF) and leukotriene B4, did not inhibit the responses of peptide chemoattractant receptors, suggesting distinct signaling pathways. To examine cross-desensitization between receptors for lipid and peptide chemoattractants, cDNA encoding the PAF receptor (PAFR) was co-expressed into RBL-2H3 cells with cDNAs encoding receptors for either formylated peptides (FR), a product of the fifth component of complement (C5aR) or interleukin-8 A (IL-8RA). PAFR was homologously phosphorylated and desensitized by PAF, and cross-phosphorylated and cross-desensitized by fMet-Leu-Phe, C5a, and IL-8. In contrast, the receptors for peptide chemoattractants were neither cross-phosphorylated nor cross-desensitized by PAF. Staurosporine blocked cross-phosphorylation and cross-desensitization of the PAFR by peptide chemoattractants. Truncation of the cytoplasmic tail of PAFR (mPAFR) abolished its homologous and cross-phosphorylation. mPAFR was also resistant to cross-desensitization by peptide chemoattractants at the level of PLC activation. Interestingly, mPAFR mediated a sustained Ca2+ mobilization in response to PAF and was more active in inducing GTPase activity, phosphoinositide hydrolysis, secretion, and phospholipase D activation than the wild type PAFR. In contrast to PAFR, stimulation of the mPAFR cross-phosphorylated and cross-desensitized responses to IL-8RA. As expected, FR, which is resistant to cross-phosphorylation by C5aR and IL-8RA, was not phosphorylated by mPAFR. However, unlike C5aR and IL-8RA, mPAFR did not inhibit the ability of FR to activate PLC. Blocking Ca2+ influx inhibited mPAFR-mediated sustained Ca2+ response, phospholipase D activation and secretion, but not phosphoinositide hydrolysis and cross-phosphorylation and cross-desensitization of IL-8RA. The data herein suggest that cross-desensitization of PAFR by peptide chemoattractants is solely due to receptor phosphorylation. The PAFR and the peptide chemoattractant receptors do not cross-regulate each other at the level of PLC, suggesting distinct regulatory pathways.

Amino Acid Sequence↗
PubMed1996

Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein activation.

To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2.

Amino Acid Sequence↗
PubMed1995

Cross-desensitization of chemoattractant receptors occurs at multiple levels. Evidence for a role for inhibition of phospholipase C activity.

To define the molecular mechanisms of cross-regulation among chemoattractant receptors, we stably coexpressed, in a rat basophilic leukemia (RBL-2H3) cell line, epitope-tagged receptors for the chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide of the fifth component of the complement system (C5a), and interleukin-8 (IL-8). All the expressed receptors underwent homologous phosphorylation and desensitization upon agonist stimulation. When co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of the other. Activation of epitope-tagged fMLP receptor (ET-FR) also cross-phosphorylated ET-C5aR and ET-IL-8RA, but ET-FR was totally resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation of [35S]guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) binding and Ca2+ mobilization were cross-desensitized by each other and by fMLP. Stimulation of [35S]GTP gamma S binding by fMLP was also not cross-desensitized by C5a or IL-8, however, Ca2+ mobilization was, suggesting a site of inhibition distal to G protein activation. Consistent with this desensitization of Ca2+ mobilization, inositol 1,4,5-trisphosphate release in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP and C5a cross-desensitized each other's ability to stimulate phosphoinositide hydrolysis. Taken together, these results indicate that receptor cross-phosphorylation correlates directly with desensitization at the level of G protein activation. The ET-FR was resistant to this process. Of note, cross-desensitization of ET-FR at the level of phosphoinositide hydrolysis and Ca2+ mobilization was demonstrated in the absence of receptor phosphorylation. This suggests a new form of chemoattractant cross-regulation at a site distal to receptor/G protein coupling, involving the activity of phospholipase C.

Amino Acid Sequence↗
PubMed1995

Regulation of human interleukin-8 receptor A: identification of a phosphorylation site involved in modulating receptor functions.

The human type A interleukin-8 receptor (IL-8RA) was modified to express an amino-terminal epitope tag and stably overexpressed in a rat basophilic leukemia cell line (RBL-2H3). This receptor (ET-IL-8RA) displayed functional properties similar to those of the native receptor in neutrophils in that exposure to IL-8 stimulated GTPase activity, phosphoinositide (PI) hydrolysis, intracellular calcium mobilization, and degranulation in a pertussis toxin (PTx) susceptible fashion. IL-8 induced dose- and time-dependent phosphorylation of ET-IL-8RA. Phorbol 12-myristate 13-acetate (PMA) treatment also resulted in phosphorylation of the receptor although to a lesser extent. Staurosporine totally blocked PMA-induced phosphorylation but only partially inhibited IL-8-mediated phosphorylation. Phosphorylation of ET-IL-8RA correlated with its desensitization as measured by GTPase activation and calcium mobilization. To determine the role of phosphorylation in IL-8RA signal transduction, three mutants lacking specific serine and threonine residues located at the C-terminal of this receptor were constructed by site-directed mutagenesis (M1, M2, and M3). The mutated receptors expressed in RBL-2H3 cells displayed pharmacological properties (Kd approximately 2-2.8 nM and Bmax approximately 3-3.5 pmol/mg of protein) similar to those of the wild-type ET-IL-8RA. M2 and M3, but not M1, showed a marked decrease in IL-8-induced phosphorylation compared to the wild-type receptor. M2 and M3 but not M1 were resistant to PMA-mediated phosphorylation and desensitization and were also more resistant to homologous desensitization than M1 or ET-IL-8RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1995

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.

Amino Acid Sequence↗
PubMed1994

Regulation of stably transfected platelet activating factor receptor in RBL-2H3 cells. Role of multiple G proteins and receptor phosphorylation.

Platelet activating factor (PAF) interacts with cell surface receptors to mediate inflammatory responses. To determine the mechanisms of PAF receptor regulation, we constructed epitope-tagged human PAF receptor cDNA (ET-PAFR) and generated stable transfectants in a rat basophilic cell line (RBL-2H3 cells). The expressed receptors displayed ligand binding and functional properties similar to the native receptors in neutrophils. PAF-stimulated intracellular Ca2+ mobilization was not inhibited by pertussis toxin (PTx), whereas phosphoinositide hydrolysis and secretion were blocked by approximately 40%. The PTx-resistant secretion mediated by PAF was, however, inhibited by guanosine 5'-O-(2-thio-diphosphate) in permeabilized RBL-2H3 cells, indicating a role for PTx-insensitive G protein. In contrast to the PAF receptor, responses mediated by formylpeptide and C5a chemoattractants were inhibited by PTx. PAF stimulated a dose- and time-dependent phosphorylation of its receptor. ET-PAFR was also phosphorylated by phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP. Staurosporine caused complete inhibition of ET-PAFR phosphorylation by PMA but only partial inhibition by PAF. Receptor phosphorylation by PAF and PMA correlated with desensitization as measured by a decrease in both PAF-stimulated GTPase activity in membranes and Ca2+ mobilization in intact cells. Phosphorylation of ET-PAFR by dibutyryl cyclic AMP was not, however, associated with desensitization. These data demonstrate that a single PAF receptor population interacts with multiple G proteins to mediate its biological responses. Moreover, ET-PAFR, unlike the formylpeptide or C5a receptors, is phosphorylated by at least three kinases (most likely protein kinases A and C and a receptor kinase). The functional consequences of cellular activation by various chemoattractants may depend upon the G protein to which their receptor is coupled.

Alkaloids↗
PubMed1993

Identification of additional members of human G-protein-coupled receptor kinase multigene family.

Human neutrophils express several distinct guanine nucleotide binding (G)-protein-coupled receptors that mediate their responsiveness to chemoattractants. Phosphorylation by receptor-specific and second messenger-activated protein kinases is a common mechanism for regulation of G-protein-coupled receptors. To explore the possibility that chemoattractant receptors are regulated by unique receptor kinases, we utilized PCR to identify receptor kinases in human neutrophils. Here, we report the isolation of three G-protein-coupled-receptor-kinase (GPRK)-like sequences termed GPRK5, GPRK6, and GPRK7 in addition to the beta-adrenergic receptor kinase (beta ARK) 1 and 2 isoforms (beta ARK1 and beta ARK2). Two, GPRK5 and GPRK6, showed high homology at the amino acid level to the recently identified receptor-kinase-like sequence localized close to the Huntington disease locus. GPRK7 is of interest in that it contains a DLG (Asp-Leu-Gly) amino acid motif of receptor kinases preceded by a DFD (Asp-Phe-Asp) motif. We isolated cDNAs corresponding to GPRK6; the complete sequence shows > 66% identity and 81% similarity at the amino acid level to the GPRK from the Huntington disease locus. The GPRK6 cDNA probe hybridizes to two mRNAs of 2.9 and 2.1 kb that were expressed in all the tested human tissues including HL-60 cells and neutrophils. Genomic Southern blot analysis and chromosome mapping showed that GPRK6 hybridizes to two closely related genes located on chromosomes 5 and 13 and are, therefore, distinct from the GPRK located near the Huntington disease locus on chromosome 4. The identification herein of three putative receptor kinases indicates that in addition to beta ARK and rhodopsin kinase subfamilies, there are other receptor-kinase subfamilies that regulate the broad spectrum of G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1991

Identification of a protein kinase multigene family of Dictyostelium discoideum: molecular cloning and expression of a cDNA encoding a developmentally regulated protein kinase.

We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs). Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family. These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases. DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2). The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids. A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast. The N-terminal domain is highly basic and also contains alternating threonine/proline residues. The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development. The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway.

Amino Acid Sequence↗
PubMed1991

Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum.

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.

Animals↗
PubMed1991

Homology cloning of protein kinase and phosphoprotein phosphatase sequences of Dictyostelium discoideum.

Reversible protein phosphorylation appears to be important at several stages in the signal transduction pathways in Dictyostelium discoideum. To elucidate its role, we have isolated sequences encoding putative protein kinases and phosphoprotein phosphatases by homology cloning using polymerase chain reactions (PCRs). Oligonucleotide primers were synthesized for use as forward and reverse primers with their nucleotide sequences deduced from the amino acid sequences of conserved domains of several protein kinases and phosphoprotein phosphatases. The fragments amplified by PCR were cloned, sequenced, and shown to encode parts of five different protein kinases and two phosphoprotein phosphatases. Several features such as the deduced amino acid sequence homology, location of invariant amino acids, GC content, and the codon usage confirmed that one set of clones encode parts of different protein kinases of Dictyostelium. Two clones derived from phosphoprotein phosphatase primers encode fragments of type 1 and type 2A phosphoprotein phosphatases. Amplified fragments were used to screen a lambda gt11 bank, and several cDNA clones for protein kinases were isolated. Some of these show differential expression during development or in response to exogenous cAMP.

Amino Acid Sequence↗
PubMed1989

Identification of a signal transduction response sequence element necessary for induction of a Dictyostelium discoideum gene by extracellular cyclic AMP.

The signal transduction pathways that lead to gene induction are being intensively investigated in Dictyostelium discoideum. We have identified by deletion and transformation analysis a sequence element necessary for induction of a gene coding for uridine diphosphoglucose pyrophosphorylase (UDPGP1) of D. discoideum in response to extracellular cyclic AMP (cAMP). This regulatory element is located 380 base pairs upstream of the transcription start site and contains a G+C-rich partially palindromic sequence. It is not required for transcription per se but is required for induction of the gene in response to the stimulus of extracellular cAMP. The cAMP response sequence is also required for induction of the gene during normal development. A second A+T-rich cis-acting region located immediately downstream of the cAMP response sequence appears to be essential for the basal level of expression of the UDPGP1 gene. The position of the cAMP response element coincides with a DNase I-hypersensitive site that is observed when the UDPGP1 gene is actively transcribed.

Base Sequence↗
PubMed1988

Transmembrane signal transduction regulates gene expression in Dictyostelium discoideum.

cAMP regulates gene expression in Dictyostelium discoideum through the cell surface receptor and is therefore a transmembrane signal transduction event. We have now begun to examine the signal transduction pathway that transmits the cAMP-induced signal to the nucleus. The results presented here indicate that Ca2+ plays a crucial role. A comparison of the accumulation of UDPGP1 mRNA during development with the corresponding transcription rates revealed that this gene is regulated primarily at the level of transcription. To elucidate the factors involved in the regulation of the UDPGP1 gene we characterized its cis acting sequences. We constructed a series of deletions into the 5' flanking region of the UDPGP1 gene and analyzed the expression of the mutated DNA in transformants. A sequence element essential for the expression of the UDPGP1 gene is located between -500 bp and -288 bp from the transcription start site. This promoter element appears to be a short G + C-rich sequence positioned between -374 to -395 and coincides with a DNase I hypersensitive site.

Base Sequence↗
PubMed1986

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum.

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.

Cell Aggregation↗
PubMed1986

Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum.

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of dictyostelium discoideum.

Uridine diphosphoglucose pyrophosphorylase (UTP: -alpha-D-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175-187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997-5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.

Cloning, Molecular↗
PubMed1985

An essential arginine residue at the substrate binding site of 4-hydroxyisophthalate hydroxylase.

4-Hydroxyisophthalate hydroxylase was inactivated by treatment with phenylglyoxal by a process obeying pseudo-first order kinetics indicating the presence of an essential arginine located presumably in the active site. Addition of saturating amounts of 4-hydroxyisophthalate during the treatment resulted in complete protection of the enzyme from the inactivation, but addition of NADPH was totally ineffective. Analysis of the effect of various substrate analogs on the protection of the enzyme showed that carboxyl and hydroxyl groups at para positions on the aromatic ring are essential for substrate binding to the active site. It was also observed that analogs which protect the enzyme against phenylglyoxal inactivation are themselves effective inhibitors of the enzyme activity.

Arginine↗