Biomedical subjects

X C Wang

Publications and source records attributed to X C Wang.

At least 55 records · Page 3Linked to original sources

PubMed1999

Suppression of NF-kappaB-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor.

PURPOSE: To determine whether nuclear transcription factor-kappaB (NF-kappaB) is activated in human retinal pigment epithelial (hRPE) cells in response to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone or in combination and if so, whether expression of proinflammatory genes induced by these agents can be blocked by a proteasome inhibitor, MG-132, which inhibits the degradation of I kappaB, an NF-kappaB inhibitor, thereby preventing nuclear translocation of NF-kappaB. METHODS: Cultured hRPE were pretreated for 60 minutes with medium alone or medium containing the proteasome inhibitor MG-132 (20 microM) and then exposed to TNF-alpha (1.1 x 10(3) U/ml), IL-1beta (5 U/ml), or IFN-gamma (7.5 x 10(3) U/ml) alone or in combination (TII). Nuclear translocation of NF-kappaB was determined by fluorescence staining of the NF-kappaB Rel A (p65) subunit. Cytoplasmic I kappaB protein was measured by western blot analysis. Nuclear extract binding to kappaB DNA motifs was measured by electrophoretic mobility shift assay and antibody supershift assay. Steady state mRNA expression of the chemokines melanoma growth stimulating activity (MGSA)/gro-alpha, regulated on activation normal T-cell expression and secreted (RANTES), and monocyte chemoattractant protein (MCP-1), the cytokines IL-1beta and macrophage colony stimulating factor (M-CSF) and intercellular adhesion molecule-1 (ICAM-1) was evaluated by semiquantitative reverse transcription-polymerase chain reaction. Chemokine and cytokine protein secretion was measured by enzyme-linked immunosorbent assay. Cell-surface ICAM-1 expression was determined by flow cytometry. RESULTS: TNF-alpha, IL-1beta, and TII but not IFN-gamma alone caused degradation of I kappaB, Rel A nuclear translocation, and increased NF-kappaB DNA binding activity, effects that were blocked by pretreatment with MG-132. MG-132 suppressed MGSA/gro-alpha, RANTES, MCP-1, IL-1beta, M-CSF, and ICAM-1 mRNA expression and secreted RANTES, MCP-1, and M-CSF protein, and cell-surface ICAM-1 that were induced by IL-1beta, TNF-alpha, and TII. CONCLUSIONS: TNF-alpha, IL-1beta, and TII induce expression of proinflammatory cytokines and ICAM-1 in hRPE cells through an NF-kappaB-dependent signal transduction pathway. This effect is blocked by MG-132, a proteasome inhibitor that prevents I kappaB degradation. Inhibition of NF-kappaB may be a useful strategy to treat proliferative vitreoretinopathy and uveitis, ocular diseases initiated and perpetuated by cytokine activation.

Blotting, Western↗

PubMed1998

Organization of the simian hemorrhagic fever virus genome and identification of the sgRNA junction sequences.

SHFV is a member of the Arteriviridae family. Viruses within this family encode eight open reading frames (ORFs), two of which are translated from the full-length genome RNA. The remaining six ORFs are translated from a nested set of six or seven 3' co-terminal, subgenomic RNAs (sgRNAs). We have cloned and sequenced approximately 6000 nucleotides (nt) from the 3' end of the SHFV genome. Eleven ORFs, numbered ORFs 1a, 1b, 2a, 2b, 3, 4, 5, 6, 7, 8, and 9, were identified, three more than the other arteriviruses. The characteristics of the peptides encoded by ORFs 2a through 9 were determined from their computer-generated amino acid sequences. We also amplified the junction sequences from each of the SHFV subgenomic RNAs (sgRNAs) using RT-PCR analysis. Eight separate junction sequences were found which suggests that SHFV produces eight sgRNAs during replication. ORFs 2a and 2b appear to be encoded on the same sgRNA implying that RNA 2 is polycistronic. Sequence analysis identified the conserved SHFV junction sequence as 5'-(U/C)(C/U)N(U/C)(U/C)(A/C/G)AC(C/U)-3'. Since SHFV encodes additional ORFs and produces additional sgRNAs during replication, these data suggest that SHFV may be more complex than the other arteriviruses.

Animals↗

PubMed1998

Chymotrypsin gene expression in rat peripheral organs.

Prior studies have revealed the presence of chymotrypsinlike protease in peripheral organs, although no definitive evidence for the synthesis of this enzyme in tissue other than the pancreas is available. In an attempt to detect chymotrypsinogen mRNA in peripheral organs, a fragment of the pancreatic chymotrypsin mRNA from rat was amplified using PCR. The sequence was identified as a portion of the rat chymotrypsin B gene overlapping exon 5 through exon 7. It was subcloned into the pGEM-4Z vector and used as a template for the vitro transcription of an antisense riboprobe. Using ribonuclease protection and Northern blot analyses, chymotrypsin mRNA was detected in the rat pancreas, stomach, duodenum, ovary, and spleen. Monoclonal and polyclonal antisera against chymotrypsin detected chymotrypsinlike immunoreactivity in rat and human pancreas, rat stomach, duodenum and jejunum. Electrophoresis and immunoblotting revealed chymotrypsin-chymotrypsinogen bands (25-29 kDa) in the stomach and duodenum. Synthesis of a potent protease such as chymotrypsin in tissue other than pancreas is significant, suggesting a potential physiological and/or pathological role in these tissues.

Animals↗

PubMed1998

Intravitreal sustained-release cyclosporine in the treatment of experimental uveitis.

OBJECTIVE: Uveitis often is a chronic disease requiring long-term medical therapy. Despite treatment, the disease may be difficult to control and may produce serious, vision-threatening ocular complications. In this study, the authors determined whether an intravitreal cyclosporine-sustained delivery device was effective in the treatment of ocular inflammation in a rabbit model of uveitis. METHODS: New Zealand White rabbits were immunized subcutaneously with Mycobacterium tuberculosis H37Ra antigen. Fourteen days later, sustained-release cyclosporine devices were implanted into the vitreous cavity of the right eye of experimental rabbits. Control animals received sham devices. Seven days after device implantation, rabbits were challenged with an intravitreal injection of tuberculin antigen. To simulate chronic inflammation with exacerbations, some animals were rechallenged with intravitreal antigen on day 21 after device implantation. Inflammation was assessed clinically by a masked observer who graded anterior chamber cells, flare, corneal neovascularization, iris congestion, and vitreous opacity daily until day 7 and on day 13 after the initial intravitreous challenge, and on days 1 and 2 after the rechallenge. Retinal function was evaluated by electroretinography. Animals were killed 3, 6, 8, and 14 days after the initial intravitreal challenge and on the second day after rechallenge for aqueous leukocyte count, protein measurement, and histologic examination. The number of aqueous and peripheral blood proliferating lymphocytes and the subset of CD4+ T cells were determined by flow cytometry. High-performance liquid chromatography was used to measure cyclosporine A levels in vitreous and peripheral blood. Light microscopy was used to evaluate the eyes histopathologically. RESULTS: By clinical criteria, treated eyes had significantly less inflammation than untreated eyes. The number of aqueous cells and protein concentration determined quantitatively paralleled the clinical assessment of anterior chamber cells and flare. The electroretinography B-wave was depressed significantly in untreated eyes compared with that of treated eyes (P < 0.02). Histopathologic examination results showed marked inflammation and tissue disorganization in untreated eyes, whereas cyclosporine-treated eyes had preserved architecture and greatly reduced inflammatory cells. Intravitreal cyclosporine remained at therapeutic levels for at least 6 months after intravitreal device implantation, whereas blood levels were low to nondetectable. CONCLUSIONS: The intravitreal cyclosporine A device effectively suppresses ocular inflammation in a rabbit model of uveitis. This device may be useful in the treatment of patients with severe chronic uveitis who are intolerant to currently available therapies.

Animals↗

PubMed1998

Purification of heparin-binding protein HBp17 and identification of HBp17 heparin binding site.

HBp17 was purified by Heparin-Copper biaffinity chromatography and HPLC from conditioned medium of A431 cell. The purified HBp17 was digested by staphylococcus urcus V8 protease or chymotrypsin and the heparin-binding fragments were isolated by Heparin-Sepharose. One binding site of peptide mapping is HBp17 residues 110-145 produced by V8. Another one is HBp17 residues 82-143 which were produced by chymotrypsin digestion. Two binding sites of peptide mapping are overlap. Therefore the residues 110-143 of HBp17 are the principle heparin binding site. The basic amino acid cluster in this region may be contribute to the binding of HBp17 to heparin or heparan sulfate proteoglycan on the cell surface and extracellular matrix.

Amino Acid Sequence↗

PubMed1998

Identification of the leader-body junctions for the viral subgenomic mRNAs and organization of the simian hemorrhagic fever virus genome: evidence for gene duplication during arterivirus evolution.

Simian hemorrhagic fever virus (SHFV) was recently reclassified and assigned to the new virus family Arteriviridae. During replication, arteriviruses produce a 3' coterminal, nested set of subgenomic mRNAs (sgRNAs). These sgRNAs arise by discontinuous transcription, and each contains a 5' leader sequence which is joined to the body of the mRNA through a conserved junction sequence. Only the 5'-most open reading frame (ORF) is believed to be transcribed from each sgRNA. The SHFV genome encodes nine ORFs that are presumed to be expressed from sgRNAs. However, reverse transcription-PCR analysis with leader- and ORF-specific primers identified only eight sgRNA species. The consensus sequence 5'-UCNUUAACC-3' was identified as the junction motif. Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and ORF 2b. SHFV encodes three more ORFs on its genome than the other arteriviruses. Comparative sequence analysis suggested that SHFV ORFs 2a, 2b, and 3 are related to ORFs 2 through 4 of the other arteriviruses. Evidence which suggests that SHFV ORFs 4 through 6 are related to ORFs 2a through 3 and may have resulted from a recombination event during virus evolution is presented.

Amino Acid Sequence↗

PubMed1997

Minimal functional unit of lactate dehydrogenase.

The tetrameric heart isozyme of lactate dehydrogenase (H4) is modified by p-chloromercuribenzoate (PCMB) to produce the inactive tetramer (H4) and then hybridized with native tetrameric muscle isozyme (M4). The hybrid mixture (M4, H'M3, H2'M2, H3'M, and H4') was isolated by polyacrylamide gel electrophoresis (PAGE) and then stained for enzyme activity and with Coomassie brilliant blue. Only three bands were found on the gels in either case. The hybrid enzymes (H'M3 and H2'M2) as isolated by PAGE have half the specific activity of the native muscle enzyme. The electrophoresis properties of H'M3 are very similar to those of HM3, while the electrophoresis properties of H2'M2 are very similar to those of H2M2. The above results strongly suggest that the tetramer having enzymatic activity contains at least two native subunits, and the di-subunit in the tetrameric enzyme is the minimal functional unit.

Animals↗

PubMed1997

Abnormal naive and memory T lymphocyte subsets in the peripheral blood of patients with uveitis.

PURPOSE: To better define the role of lymphocytes in the pathogenesis of uveitis, we studied the expression of memory and naive cell markers on T lymphocytes from peripheral blood. METHODS: Surface antigens on T lymphocytes obtained from peripheral blood of 27 patients with uveitis, including 12 patients with Behçet's disease (BD), 7 patients with Vogt-Koyanagi-Harada disease (VKH), and 8 patients with idiopathic uveitis (IU), were detected by three-color flow cytometric analysis. Lymphocytes from 14 age-matched healthy control subjects were similarly evaluated. RESULTS: The percentage of T lymphocytes that were CD4+CD29+ lymphocytes (memory cells) was high in all patients with uveitis, while that of CD4+CD45RA+ lymphocytes (naive cells) was lower in patients with BD and VKH, although the difference was not statistically significant. The percentage of CD29+ cells within CD3+CD4+ cell population was significantly higher in patients with BD and VKH than in the controls (p < 0.01), and the percentage of CD45RA+ cells was significantly lower in BD patients than in controls (p < 0.01). The T lymphocyte subsets in patients with IU were similar to the controls. CONCLUSIONS: These results show an abnormal distribution of T lymphocytes in patients with uveitis associated with an underlying systemic disease.

Adult↗

PubMed1997

Cytoplasmic creatine kinases from giant pandas.

The muscle and brain creatine kinases of giant panda have been isolated and purified. The purified muscle and brain enzymes (MM and BB) are homogeneous on both the polyacrylamide gel electrophoresis in the presence and absence of SDS. Both enzymes are dimers, consisting of two identical subunits each with a molecular weight of 42,000 daltons. The characteristics of muscle and brain enzymes have been studied, respectively. The hybridized enzyme, MB, was prepared by hybridization of MM and BB. The kinetic parameters of MM, BB and MB were determined, respectively. The results from modification of SH groups show that the SH groups of panda creatine kinases are essential for their activity and among the all SH groups in the enzyme only one per subunit is essential for enzymatic activity.

Amino Acids↗

PubMed1997

Connexin domains relevant to the chemical gating of gap junction channels.

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with asparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL (IL1) and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively.

Animals↗

PubMed1996

The genomic structure of discoidin receptor tyrosine kinase.

The discoidin domain receptor (DDR) is a new class of receptor tyrosine kinase that is distinguished by a unique extracellular domain homologous to the lectin Discoidin I found Dictyostelium discoideum. A cosmid was isolated from a human chromosome 6 cosmid library containing the DDR gene. A complete genomic contig of the DDR gene was constructed from seven subclones of the cosmid. The cosmid fragments were analyzed by PCR, sequencing, and comparison of genomic/cDNA sequence. The DDR gene is composed of 17 exons, ranging in size from 96 to 1014 bp, distributed along approximately 12 kb of genomic DNA. The extracellular domain is encoded by 8 exons of which three code for the discoidin domain. The transmembrane domain is encoded by 1 exon, the juxtamembrane by 3 exons, and the catalytic domain by 5 exons. The generation of the two splice variants of DDR, EDDR1 and EDDR2 are explained by the genomic structure. Exon 11 (111 bp in the juxtamembrane domain) is present in DDR and absent in the splice variant EDDR1. An inverted repeat of 20 bp was identified at the 3' exon-intron junction of exon 11, which results in a lariat loop-like secondary structure. EDDR2 is generated because of a cryptic splice acceptor site that results in an extra 18 bp (6 amino acids) inserted 5' of exon 14 in the catalytic domain. A polymorphic (GT)17 repeat was identified in intron 5 with a heterozygosity of 0.71. The exon-intron structure of the DDR gene will be helpful in further understanding of its function and explains the possible structural basis for the two splice variants.

Alternative Splicing↗

PubMed1996

Dissociation, unfolding and inactivation of creatine kinase in urea solutions.

It was previously reported that, during unfolding of creatine kinase in guanidinium chloride or urea solutions, inactivation occurred before noticeable conformational change could be detected, suggesting that the conformation at the active site is more easily perturbed and, hence, more flexible than the molecule as a whole [Tsou (1986) Trends Biochem. Sci 11, 427-429]. In the present paper, the ureagradient electrophoresis and the isoenzyme hybrid of creatine kinase has been studied. The results show that at low urea concentrations, creatine kinase is still in the dimeric state or only slightly dissociated. The dissociation and inactivation of creatine kinase during denaturation by urea are also compared. It was found that the enzyme was nearly inactivated in low urea concentrations before noticeable dissociation was detected. It therefore appears that in low urea concentrations, inactivation of creatine kinase is not due to the dissociation of the active dimer. The present result supports the hypothesis of the conformational flexibility of the active site in this enzyme.

Animals↗

PubMed1996

Apoptosis-related fas antigen on memory T cells in aqueous humor of uveitis patients.

To investigate the role of lymphocytes in the pathogenesis of uveitis, we analyzed the expression of memory markers, CD29 and CD45RO antigens, and apoptosis-related Fas antigen on T lymphocytes in the aqueous humor (AH) and peripheral blood (PB) from patients with uveitis. Using three-color flow cytometry, we assessed the number of T lymphocyte subsets that stained with fluorescence-conjugated anti-CD3, CD4, CD8, CD29, CD45RA, CD45RO, HLA-DR, and Fas monoclonal antibodies in the AH and PB from 19 patients with active uveitis who were diagnosed as having sarcoidosis, Vogt-Koyanagi-Harada disease, HLA-B27+ uveitis, or idiopathic uveitis. Cells from AH and PB were evaluated by light and electron microscopy before and after 6 h of incubation. The majority of lymphocytes in AH but not in PB, were CD3+HLA-DR+ (activated) T cells. The percentage of CD4+ lymphocytes was significantly higher in uveitic AH than in PBL (P < 0.01). While the percentage of CD4+ CD45RA+ (naive) cells within T cells was much lower in uveitic AH than in PB, the percentage of CD4+CD29+ or CD4+CD45RO+ (memory) cells was significantly higher in uveitic AH than in PBL (P < 0.01). Fas antigen was expressed preferentially on memory cells in uveitic AH. Apoptosis of cells in the AH was observed by microscopically following after incubation with no stimulation. Lymphocytes from the AH of patients with uveitis were more activated than those from PB. The majority of T lymphocytes from uveitic AH expressed memory markers and Fas antigen. Results suggest that an increase in the number of Fas+ memory T lymphocytes in AH is involved in the pathogenesis of uveitis.

Adolescent↗

PubMed1996

H-RYK, an unusual receptor kinase: isolation and analysis of expression in ovarian cancer.

BACKGROUND: Protein tyrosine kinases play an important role in cellular metabolism as key components of signal transduction pathways. They are involved in cellular growth, differentiation, and development. Receptor tyrosine kinases (EGF receptor and c-erbB2) have been shown to be important in the pathogenesis of cancer. In ovarian cancer, overexpression of c-erbB2, a type I receptor, has been correlated with an adverse effect on survival of patients. MATERIAL AND METHODS: An unusual receptor tyrosine kinase, H-RYK, has been isolated from a complimentary DNA library of SKOV-3, an epithelial ovarian cancer cell line, using a polymerase chain reaction-mediated approach. RESULTS: The primary structure of the predicted amino acid sequence of the protein shows a novel NH2-terminal region. The catalytic region shows homology to other tyrosine kinases, the closest homology being with v-sea (39%). A significant alteration in the catalytic domain is that the highly conserved "DFG" triplet in subdomain VII is altered to "DNA." The gene was mapped to chromosome 3q22. A single transcript of 3.0 kb is expressed in heart, brain, lung, placenta, liver, muscle, kidney, and pancreas by Northern analysis with maximal expression in skeletal muscle. In situ hybridization analysis on human tissues demonstrated localization of message in the epithelial and stromal compartment of tissues such as brain, lung, colon, kidney, and breast. There was minimal to absent expression of H-RYK on surface epithelium of ovaries. In benign (3) and borderline tumors of the ovary (5), there was expression in the stromal compartment. However, in malignant tumors (24) there was increased expression predominantly confined to the epithelium. Polyclonal antisera raised against synthetic peptides recognize a 100-kD protein in ovarian cancer cells and other cell lines. In contrast to other receptor tyrosine kinases, the receptor did not phosphorylate in an in vitro kinase assay. CONCLUSIONS: The expression of this unusual receptor tyrosine kinase in epithelial ovarian cancer suggests that it may be involved in tumor progression, which needs further investigation.

Adenocarcinoma↗

PubMed1995

Two-color flow cytometric analysis of activated T lymphocytes in aqueous humor of patients with endogenous vs. exogenous uveitis.

To investigate the immunopathologic role of lymphocytes in aqueous humor of patients with uveitis, we assessed activated T lymphocyte subsets stained with fluorescein isothiocyanate-labeled and phycoerythrin-labeled monoclonal antibodies in aqueous humor and in peripheral blood, using two-color flow cytometry. We divided our 25 patients into those with endogenous or exogenous uveitis. The percentages of activated T lymphocytes in uveitic aqueous humor significantly exceeded those in peripheral blood lymphocytes from patients with either disease form. CD4+HLA-DR+ lymphocytes were predominant in endogenous uveitic aqueous humor, whereas CD8+HLA-DR+ lymphocytes predominated in exogenous uveitic aqueous humor in the early stage of the disease. The percentages of CD4+HLA-DR+ lymphocytes tended to increase while those of CD8+HLA-DR+ cells tended to decrease in aqueous humor from patients in the late phase of exogenous uveitis. Our results may contribute to increasing diagnostic accuracy in patients with uveitis and to developing more rational methods of treatment.

Adult↗

PubMed1995

Effect of succinyl cholecystokinin heptapeptide on synaptosomal 45Ca uptake of mouse hippocampus.

AIM: To study the effect of a new peptide, succinyl cholecystokinin heptapeptide (SCH), on the uptake of 45Ca by synaptosomes from mouse hippocampus, and compare it with that of sincalide. METHODS: Protein concentration was determined by the dye method. 45Ca uptake was determined by adding 45CaCl2 into synaptosome suspending solution. Samples were incubated and terminated by filtration through glass microfiber filters and finally counted in a Beckman LS 9800 liquid scintillation spectrometer. RESULTS: SCH 0.125 - 2 mumol.L(-1) dose-dependently depressed synaptosomal 45Ca uptake, and the depressive extent was similar to that of sincalide. The suppressive effect induced by either SCH or sincalide on synaptosomal 45Ca uptake was antagonized by proglumide, which itself did not affect 45Ca uptake of synaptosomes. The inhibition of synaptosomal 45Ca uptake induced by SCH or sincalide was blocked by beta-endorphin. CONCLUSION: The replacement of the N-terminal aspartic acid residue of sincalide does not influence its effect on 45Ca uptake of hippocampal synaptosomes.

Animals↗

PubMed1995

Ocular findings in a patient with Prader-Willi syndrome.

A 25-year-old woman is described whose clinical features included infantile hypotonia, obesity after infancy, intellectual impairment, dysmorphic facial features, short stature, small hands and feet, and abnormal dentition, which are typical of the Prader-Willi syndrome. The patient had almond-shaped eyes, spot-like hypopigmentation under the retina, and a polychromatic luster in the anterior and posterior subcapsular regions of both lenses. Using fluorescence fundus angiography, we identified choroid-transmitted fluorescence in the areas of spot-like hypopigmentation. Recordings of both electroretinogram and visual evoked potential were normal. Ophthalmologists should be aware of the characteristic features of Prader-Willi syndrome because some of the ocular disorders associated with this syndrome can be treated.

Adult↗

PubMed1994

Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination.

Whole leaves and guard-cell protoplasts of the C3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), Vmax and Km(PEP.Mg), were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mM glucose-6-Pfree (Glc-6-P, an activator), and the presence of 5 mM malatefree (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or--and less likely--that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically. The values of Vmax were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vmax were restricted to a small range (52.4 +/- 5.9 (SD) to 64.4 +/- 4.8 (SD) mumol.g fresh mass-1.h-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC Vmax was also restricted to a small range: 7.48 +/- 0.89 (SD) pmol.guard-cell pair-1.h-1 (pH 8.1, light, +Glc-6-P) to 5.79 +/- 0.60 (SD) pmol.guard-cell pair-1.h-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in Km(PEP.Mg) were calculated (whole-leaf PEPC: 0.03 to 3.84 mM; guard-cell PEPC: 0.06 to 3.43 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Fabaceae↗