Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 1,027 records · Page 57Linked to original sources

PubMed1998

Duplication of the DR3 gene on human chromosome 1p36 and its deletion in human neuroblastoma.

The human DR3 gene, whose product is also known as Wsl-1/APO-3/TRAMP/LARD, encodes a tumor necrosis factor-related receptor that is expressed primarily on the surface of thymocytes and lymphocytes. DR3 is capable of inducing both NF-kappa B activation and apoptosis when overexpressed in mammalian cells, although its ligand has not yet been identified. We report here that the DR3 gene locus is tandemly duplicated on human chromosome band 1p36.2-p36.3 and that these genes are hemizygously deleted and/or translocated to another chromosome in neuroblastoma (NB) cell lines with amplified MYCN. Duplication of at least a portion of the DR3 gene, including the extracellular and transmembrane regions but not the cytoplasmic domain, was demonstrated by both fluorescence in situ hybridization and genomic Southern blotting. In most NB cell lines, both the DR3 and the DR3L sequences are simultaneously deleted and/or translocated to another chromosome. Finally, DR3/ Wsl-1 protein expression is quite variable among these NB cell lines, with very low or undetectable levels in 7 of 17 NB cell lines.

Animals↗

PubMed1998

Evidence that HetR protein is an unusual serine-type protease.

The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria. To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli. Purified rHetR was unstable and degraded easily in solution. Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR. Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca2+. S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced. The labeled peptide matches a sequence from HetR. These results show that HetR is a protease.

Amino Acid Sequence↗

PubMed1998

Are protein folds atypical?

Protein structures are a very special class among all possible structures. It has been suggested that a "designability principle" plays a crucial role in nature's selection of protein sequences and structures. Here, we provide a theoretical base for such a selection principle, using a simple model of protein folding based on hydrophobic interactions. A structure is reduced to a string of 0s and 1s, which represent the surface and core sites, respectively, as the backbone is traced. Each structure is therefore associated with one point in a high dimensional space. Sequences are represented by strings of their hydrophobicities and thus can be mapped into the same space. A sequence that lies closer to a particular structure in this space than to any other structures will have that structure as its ground state. Atypical structures, namely those far away from other structures in the high dimensional space, have more sequences that fold into them and are thermodynamically more stable. We argue that the most common folds of proteins are the most atypical in the space of possible structures.

Models, Biological↗

PubMed1998

Adenosine A2 receptor mediation of pre- and postsynaptic excitatory effects of adenosine in rat hippocampus in vitro.

Excitatory effects of adenosine in the rat hippocampus were studied by intracellular recording from CA1 pyramidal cells in vitro. Application of 100 microM adenosine induced a rapid hyperpolarization and a decrease in input resistance, and depressed the excitatory postsynaptic potentials (EPSPs) evoked by stimulation of Schaffer collaterals in all neurons tested. In 55% of the neurons this was followed by an excitation. This excitation consisted of a slow depolarization lasting 9 +/- 4.7 min, an increase in input resistance and an increase in the amplitude of the evoked EPSPs. This excitation could also be observed when synaptic transmission was prevented by 1 microM tetrodotoxin (tetrodotoxin). In the presence of 5 microM 8-(p-sulphophenyl)theophylline (8-SPT) adenosine (10 microM) enhanced the amplitude of evoked EPSPs by 20% +/- 3.6 (n = 5) in all neurons tested. Chloroadenosine (chloroadenosine; a stable analog of adenosine; 20 microM) induced a similar hyperpolarization associated with decrease in input resistance; this was followed by a similar excitation as seen with adenosine in 22 of the 27 neurons tested. L-baclofen (20 microM) induced a hyperpolarization associated with decrease in input resistance in all six neurons tested but an ensuing excitation was not observed. CGS 21680 (30 nM), a selective adenosine A2 receptor agonist, induced a slow depolarization associated with an increase in input resistance and, in 11 of 36 neurons, enhanced the amplitude of evoked EPSPs. These excitatory effects of CGS 21680 were blocked by the selective adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 10 microM). In the presence of 1 microM tetrodotoxin 30 nM CGS 21680 still produced a slow depolarization and an increase in input resistance. In addition, high doses of CGS 21680 (10 and 20 microM) depressed the amplitude of EPSPs evoked by stimulation of Schaffer collateral afferents, yet there was little effect on the resting membrane potential or input resistance despite the fact that 20 microM chloroadenosine caused a pronounced hyperpolarization associated with a decrease in input resistance in the same cell. These results indicate that the excitatory effects of adenosine may be mediated via activation of adenosine A2 receptors at both presynaptic and postsynaptic sites in the hippocampal CA1 region.

2-Chloroadenosine↗

PubMed1998

The N terminus of eukaryotic translation elongation factor 3 interacts with 18 S rRNA and 80 S ribosomes.

Elongation factor-3 (EF-3) is an essential fungal-specific translation factor which exhibits a strong ribosome-dependent ATPase activity and has sequence homologies that may predict domains critical for its role in protein synthesis, including a domain at the N terminus, which exhibits sequence homology with Escherichia coli ribosomal protein S5. A portion of the N terminus of Saccharomyces cerevisiae EF-3 (spanning the S5 homology region) has been cloned, expressed, and purified from E. coli. UV cross-linking experiments revealed that the N-terminal EF-3 protein (N-term EF-3) can be specifically cross-linked to 18 S rRNA. Filter-binding assays confirmed these data, and also established that the interaction has a Kd approximately 238 nM. Additional evidence shows that N-term EF-3 is able to associate with yeast ribosomes and inhibit the ribosome-dependent ATPase activity of native EF-3. These data taken together suggest that at least one of the ribosome-binding sites of EF-3 is located at the N terminus.

Cloning, Molecular↗

PubMed1998

Functional implications of the 21-24 loop in recombinant prochymosin.

To investigate the role of the 21-24 (pepsin numbering) loop in prochymosin, the amino acid residues GTPP at positions 21 through 24 were replaced with GG, the equivalent loop residues from its homologous protein, penicillopepsin, or SG, GS by site-directed mutagenesis. The mutants except GTPP(21-24)GS could be expressed in Escherichia coli. Activation studies indicated that the refolded prochymosin mutants were capable of undergoing autocatalytic activation to produce pseudochymosin by cleaving its N-terminal 27 amino acid residues at pH 2. The resulting pseudochymosin mutants were able to convert into chymosin at pH 5.5 by further autocatalytic cleavage to remove additional 15 amino acid residues. These results demonstrate that the prochymosin analogs can fold into an active state from an unfolded state and that the pseudochymosin analogs can proceed in the transformation from one active form into another active form. Spectroscopic analyses revealed that after mutation the far UV CD spectrum of prochymosin was considerably modified, showing less negative ellipticity values, and the fluorescence emission intensities of prochymosin and pseudochymosin were remarkably reduced. The stabilities of prochymosin and pseudochymosin, especially, were dramatically decreased. The stabilization energy of prochymosin was reduced by 7-8 kJ/mol. The inactivation temperature of pseudochymosin was decreased by 15-20 degrees C. The wild-type pseudochymosin was stable at pH 1.5 and 6.5, whereas the mutants were completely inactivated at the same pH values. Taken together, it is reasonable to conclude that the 21-24 loop (GTPP) plays an important role in determining the stability of prochymosin and pseudochymosin, although the mutants with mutated loop (GG or SG) still can refold into an active conformation.

Animals↗

PubMed1998

Crystal structure and evolution of a transfer RNA splicing enzyme.

The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

Amino Acid Sequence↗

PubMed1998

New mutations in the KVLQT1 potassium channel that cause long-QT syndrome.

BACKGROUND: Long-QT syndrome (LQTS) is an inherited cardiac arrhythmia that causes sudden death in young, otherwise healthy people. Four genes for LQTS have been mapped to chromosome 11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3), and 4q25-27 (LQT4). Genes responsible for LQT1, LQT2, and LQT3 have been identified as cardiac potassium channel genes (KVLQT1, HERG) and the cardiac sodium channel gene (SCN5A). METHODS AND RESULTS: After studying 115 families with LQTS, we used single-strand conformation polymorphism (SSCP) and DNA sequence analysis to identify mutations in the cardiac potassium channel gene, KVLQT1. Affected members of seven LQTS families were found to have new, previously unidentified mutations, including two identical missense mutations, four identical splicing mutations, and one 3-bp deletion. An identical splicing mutation was identified in affected members of four unrelated families (one Italian, one Irish, and two American), leading to an alternatively spliced form of KVLQT1. The 3-bp deletion arose de novo and occurs at an exon-intron boundary. This results in a single base deletion in the KVLQT1 cDNA sequence and alters splicing, leading to the truncation of KVLQT1 protein. CONCLUSIONS: We have identified LQTS-causing mutations of KVLQT1 in seven families. Five KVLQT1 mutations cause the truncation of KVLQT1 protein. These data further confirm that KVLQT1 mutations cause LQTS. The location and character of these mutations expand the types of mutation, confirm a mutational hot spot, and suggest that they act through a loss-of-function mechanism or a dominant-negative mechanism.

Case-Control Studies↗

PubMed1998

A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.

Amino Acid Sequence↗

PubMed1998

NO news is good news.

The recent determination of the crystal structure of microsomal cytochrome P450 reductase, a diflavin protein that shuttles electrons from NADPH to the P450 heme, represents a significant advance towards the understanding of cytochromes P450. A similar advance was made in a related enzyme system, nitric oxide synthase (NOS). The crystal structure of the NOS heme domain reveals a very different architecture to that observed in P450s and offers significant insight into the production of nitric oxide, one of nature's most important regulatory molecules.

Binding Sites↗

PubMed1998

Beta3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells.

BACKGROUND: Treatment with an antibody that binds beta3 integrins (abciximab; c7E3 Fab) at the time of coronary angioplasty decreases the need for repeat revascularization. Two potential mechanisms have been proposed to explain this effect: (1) inhibition of platelet aggregation or (2) interruption of ligand binding to beta3 integrins on the smooth muscle cell (SMC) surface. We examined the latter hypothesis by determining (1) if beta3 integrin expression is upregulated after vascular injury in the baboon, (2) if 7E3 binds beta3 integrins on cultured SMC, and (3) if beta3 integrin activation plays a role in proliferation of cultured SMC. METHODS AND RESULTS: Results demonstrated that immunostaining for beta3 integrins was present in the neointima 1 week after balloon withdrawal injury of baboon brachial arteries and that beta3 integrin expression colocalized with alpha-actin-positive cells. In contrast, staining for beta3 integrins was undetectable in contralateral uninjured brachial arteries. 7E3 bound to cultured human aortic SMC with an affinity (KD=3.3 nmol/L) similar to 7E3 binding to endothelial cells or platelets. Cotreatment with 7E3 partially inhibited thrombospondin-induced or alpha-thrombin-induced proliferation but not PDGF-induced or serum-induced proliferation. CONCLUSIONS: In summary, these studies demonstrate that vascular cell beta3 integrin expression is increased after injury, that 7E3 binds to cultured SMC with high affinity, and that beta3 activation is important for thrombospondin-induced or alpha-thrombin-induced proliferation. These results support the hypothesis that beta3 integrins play a role in SMC growth responses after balloon injury.

Abciximab↗

PubMed1998

The receptor-associated coactivator 3 activates transcription through CREB-binding protein recruitment and autoregulation.

Transcriptional coactivators are involved in gene activation by nuclear hormone receptors. The receptor-associated coactivator 3 (RAC3) was recently identified to be highly related to the steroid receptor coactivator-1 and transcriptional intermediate factor 2, thereby establishing a novel family of nuclear receptor coactivators. In this study, we identified a RAC3 fragment containing three LXXLL motifs conserved among this family, which is sufficient to mediate nuclear receptor interaction in vivo and in vitro. Point mutations that disrupt ligand-dependent activation function of the receptor inhibited the interaction. We found that a 162-amino acid fragment of RAC3 conferred transcriptional activation and recruited the CREB-binding protein and that three distinct LXXLL motifs mediated the transcriptional activation. A trimeric far Western analysis demonstrated the formation of a ternary complex containing CREB-binding protein, RAC3, and the receptor. In addition, we showed that RAC3, transcriptional intermediate factor 2, and steroid receptor coactivator-1 are expressed in specific tissues and cancer cells and that RAC3 transcript is directly up-regulated by retinoid treatment. These results suggest that RAC3 may contribute to amplified transcriptional responses through both recruitment of additional coactivators and autoregulation by the receptor-coactivator complex.

Animals↗

PubMed1998

Bidirectional synaptic plasticity in the rat basolateral amygdala: characterization of an activity-dependent switch sensitive to the presynaptic metabotropic glutamate receptor antagonist 2S-alpha-ethylglutamic acid.

This study examines forms of activity-dependent synaptic plasticity in the basolateral amygdala in vitro and demonstrates that a brief high frequency stimulus (HFS) train can induce a switch in the direction of the enduring change in synaptic strength induced by subsequent low-frequency stimulation (LFS). LFS (1 Hz, 15 min) of the external capsule (EC) induced a persistent 1.7-fold enhancement in the amplitude of synaptic potentials recorded intracellularly in basolateral amygdala neurons. The enhancement occurred gradually during the stimulation and was maintained for >30 min after termination of the stimulus train. LFS-induced enduring synaptic facilitation was not affected by the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoate (APV; 100 microM). Brief high-frequency EC stimulation (HFS; 100 Hz, 1 sec) induced APV-sensitive short-term potentiation (2.5-fold) that generally decayed within 10 min. When LFS was applied after recovery from the short-term potentiating effect of HFS (HFS/LFS), there was an initial transient (<10 min) enhancement of the synaptic response followed by persistent synaptic depression (synaptic potential amplitude reduced by 22% at 30 min). This represents the first demonstration of stimulus-dependent long-lasting synaptic depression in the amygdala. Application of the presynaptic (group II) metabotropic glutamate receptor antagonist 2S-alpha-ethylglutamic acid (EGLU; 50 microM) prevented the HFS-dependent switch from synaptic facilitation to depression. Thus, LFS in the in vitro amygdala slice can induce either enduring synaptic potentiation or depression, depending on whether a priming HFS train has been applied. This experience-dependent switch, a novel form of metaplasticity, is not dependent on NMDA receptors but may require group II metabotropic glutamate receptors. In the amygdala, experiential modification of activity-dependent long-term synaptic plasticity adds flexibility to the ways in which synaptic strength can be modified and could play a role in diverse amygdala-dependent processes, including the formation, storage, and extinction of emotional memory and the regulation of epileptogenesis.

Amygdala↗

PubMed1998

Sulfate activation and transport in mammals: system components and mechanisms.

Extensive studies on the mammalian sulfate-activating enzymes and PAPS translocase have enhanced our understanding of the overall pathway of sulfate activation and utilization. Isolation of the PAPS-synthesizing activities from rat chondrosarcoma and preparation of stable non-hydrolyzable analogs of APS and PAPS have facilitated the kinetic characterization of mammalian ATP sulfurylase and APS kinase. These studies provided the basis for further experimental work showing that APS, the labile intermediate product, is channeled directly between the sulfurylase and kinase active sites. The defect in the brachymorphic mutant mouse lies in this channeling mechanism, thus interfering with efficient PAPS production. The rat chondrosarcoma ATP sulfurylase and APS kinase activities, in fact, reside in a single bifunctional cytoplasmic protein, which has now been cloned and expressed. The mechanism by which PAPS reaches its sites of utilization in the Golgi lumen has also been elucidated: The PAPS translocase is a 230-kDa integral Golgi membrane protein which functions as an antiport.

Animals↗

PubMed1998

Apoptotic killing of CD4+ T lymphocytes in HIV-1-infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells.

In vitro infection of PHA-stimulated, normal CD4+ human peripheral blood T lymphocytes (PBLs) with several HIV-1 isolates did not result in cytopathology, despite high levels of virus replication and the fact that some of these isolates were cytopathic in certain cell lines. In contrast, infection of unfractionated PBLs (containing CD8+ as well as CD4+ lymphocytes) with these isolates always resulted in death of the infected CD4+ T lymphocytes. It has been well documented that PHA stimulation and culture of PBLs in medium containing IL-2 generates lymphokine-activated killer (LAK) cell activity which can destroy many transformed cells and virus-infected normal cells. When CD8+ T lymphocytes from PHA-stimulated PBLs were added to HIV-1-infected purified CD4+ T lymphocytes, significant lysis occurred. This cytotoxicity was not MHC class I-restricted, and depletion of CD8+ T lymphocytes from unfractionated PBL cultures shortly after HIV infection largely abolished the killing of the infected CD4+ T lymphocytes. These results demonstrated that CD8+ LAK cells were killing the CD4+ T lymphocytes in unfractionated PBL cultures infected with these noncytopathic HIV-1 strains. Care is thus warranted when studying HIV cytopathology in unfractionated PBL cultures. Morphological and DNA gel electrophoretic analyses of HIV-infected CD4+ T lymphocytes being killed by CD8+ LAK cells demonstrated that apoptosis was the predominant mechanism of LAK cell-mediated killing. In contrast, necrosis was the major mechanism involved in killing of purified CD4+ T lymphocytes by HIV-1 strains which were directly cytopathic. These findings may explain some of the discrepancies in the literature concerning reports of either apoptotic or necrotic killing of cells by HIV in vitro. Moreover, these data strongly suggest that direct killing by replicating HIV-1 in vivo should reveal necrotic cells and immune effector cell killing should reveal apoptotic cells. Since the latter are much more frequently observed in vivo, perhaps immune effector-mediated depletion of CD4+ T lymphocytes is more important as a pathogenic mechanism.

Apoptosis↗

PubMed1998

Effect of calcium on azoxymethane-induced aberrant crypt foci and cell proliferation in the colon of rats.

Calcium has been proposed for prevention of colon cancer. The effects of calcium on azoxymethane (AOM)-induced aberrant crypt foci (ACP), a putative precancerous lesion, and cell proliferation were determined in rat colon. Male F344 rats were given AIN-76A diet that contained calcium at concentrations of 0.5, 1.0, 2.5, 5.0, 10.0 and 15.0 g/kg in experiment 1 and 0.2, 2.0 and 6.0 g/kg in experiment 2. One week after the rats received these diets, they were given the first of two weekly 15 mg/kg injections of AOM. The rats were killed after 35 days of exposure to the different diets. In experiment 1, exposure to either low (0.5 and 1.0 g/kg) or high (10.0 and 15.0 g/kg) concentrations of calcium reduced the yield of ACF relative to 5.0 g/kg calcium. In experiment 2, exposure to 0.2 and 2.0 g/kg calcium resulted in a lower yield of ACF than 6.0 g/kg. Cell proliferation in ACF and non-involved crypts was reduced in animals that received 0.5 or 15.0 mg/Kg relative to 5.0 mg/kg diet calcium. Our results indicate that both lower and higher concentrations of calcium relative to its standard concentration in AIN-76A diet can prevent ACF and reduce the extent of cell proliferation in the lesion which would likely lead to the prevention of colon cancer.

Animals↗

PubMed1998

[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions.

Human serum transferrin (hTF) is a single-chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo-protein, binds strongly to transferrin receptors and is taken up by cells via receptor-mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant epsilon-[13C]Met-hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met-464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C-lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.

Aluminum↗

PubMed1998

Interdomain information transfer during substrate activation of yeast pyruvate decarboxylase: the interaction between cysteine 221 and histidine 92.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at two cysteines on the beta domain (221 and 222) and at H92 on the alpha domain, across the domain divide from C221. While C221 has been shown to provide the trigger for substrate activation [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635], the information must be transmitted from the substrate bound at this site [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600] to the active center thiamin diphosphate located at the interface of the alpha and gamma domains. Substitution at H92 with G, A, or C leads to great reduction of the Hill coefficient (from 2.0 in the wild-type enzyme to 1.2-1.3), while substitution for Lys affords an active enzyme with a Hill coefficient of 1.5-1.6. Iodoacetate at 10 mM reduced the Hill coefficient from 2.0 to 1.1, while also causing significant inactivation of the enzyme, presumably by carboxymethylation of C221. 1,3-Dibromoacetone, a potential cross-linker when added to the H92C/C222S variant at 0.1 mM, abolished substrate activation while reducing the activity only by 30%. Therefore, 1,3-dibromoacetone may cross-link C92 and C221. It was concluded that H92 is on the information transfer pathway during the substrate activation process and the interaction between C221 on the beta domain and H92 on the alpha domain is required for substrate activation. Extensive pH studies of the steady-state kinetic constants provide support for the interaction of C221 and H92 and the transmission of regulatory information to the active center via this pathway and pKaS for the two groups. This important interaction between the C221-bound pyruvate and His92 probably has both electrostatic and steric components.

Cysteine↗