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Biomedical subjects

H Wong

Publications and source records attributed to H Wong.

At least 127 records · Page 7Linked to original sources
PubMed1995

Pulmonary responses to purified zinc oxide fume.

BACKGROUND: Metal fume fever is a flu-like illness caused by zinc oxide fume inhalation and mediated by unknown mechanisms. It is one of a group of work-related febrile inhalational syndromes. We studied bronchoalveolar lavage (BAL) obtained from cigarette smoking and nonsmoking human volunteers after controlled exposure to purified zinc oxide fume to explore the possible roles of proinflammatory cytokines in this condition. METHODS: We studied 14 volunteers after inhalation exposure to purified zinc oxide fume and after sham exposure to air. The mean cumulative exposure was 537 +/- 232 mg min per cubic meter elemental zinc. Twenty hours after exposure we performed BAL. We analyzed BAL cells and studied BAL supernatant for cytokines including tumor necrosis factor-alpha (TNF alpha), interleukin(IL)-8, and IL-1 by enzyme-linked immunosorbant assay (ELISA). RESULTS: Polymorphonuclear leukocytes (PMNs) were significantly increased in the BAL fluid obtained post-exposure compared to sham (mean difference = 41.3 +/- 16.8 x 10(3) per mL; p < 0.05). Cumulative zinc exposure positively correlated with exposure-sham differences in BAL supernatant concentrations of both TNF (r2 = 0.58; p = .002) and IL-8 (r2 = 0.44, p = 0.01). Exposure-sham concentration differences in BAL supernatant IL-8 and BAL PMNs were also positively correlated (r2 = 0.60; p < 0.001). Cigarette smoking was not associated with exposure-sham differences in BAL TNF or IL-8, but did demonstrate a packs-per-day dependent increase in BAL supernatant IL-1 (t = 2.3, p = 0.04) post-exposure compared to sham, after taking into account the zinc exposure response. CONCLUSIONS: Purified zinc oxide fume inhalation causes an exposure-dependent increase in proinflammatory cytokines and PMNs in the lung. This supports a role for cytokine networking in mediating metal fume fever.

Adult↗
PubMed1995

Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase.

Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization.

Alkaline Phosphatase↗
PubMed1994

Lipoprotein lipase domain function.

Human lipoprotein lipase (LPL) monomer consists of two domains, a larger NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a major determinant of heparin binding. Analyses of NH2-terminal domain function were performed after site-directed mutagenesis of the putative active-site serine residue, while COOH-terminal domain function was assessed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cysteine, or glycine were transiently expressed in COS-7 cells. Mutant proteins were synthesized and secreted at levels comparable to native LPL; however, none of the mutants retained enzymatic activity. The mutant with alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable to native LPL. These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of the smaller COOH-terminal domain, LPL lipolytic and esterolytic activities as well as heparin binding properties were determined after reaction with a monoclonal antibody specific for this domain. Lipolytic activity was inhibited by the monoclonal antibody, whereas esterolytic activity was only marginally affected, indicating that the LPL COOH-terminal domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, suggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) binding of antibody did not cause dimer dissociation. A model is proposed for the two LPL domains fulfilling different roles in the lipolytic process.

Animals↗
PubMed1994

Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method.

We have developed a method to monitor mRNA expression that is based upon the reverse transcriptase-polymerase chain reaction (RT-PCR) and includes multiple sets of primer pairs in coamplification reactions. To observe relative changes in mRNA steady-state levels, each target in a multiplex reaction was amplified to within a predetermined range by using PCR cycle numbers specific for each target. Optimal PCR cycle numbers for target templates were determined by preliminary titration experiments performed using the "primer-dropping" method. By employing this method, the overall amplification reaction was limited, permitting the PCR products to remain within the exponential range of the amplification curve and yet be detectable on ethidium bromide-stained gels. We demonstrated the utility of this method by monitoring the expression kinetics of cyclins A, B1, D1, and E, and of the immediate-early genes c-fos, c-myc, and beta-actin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the multiplex reactions as an endogenous internal standard to control for variations in product abundances due to differences in individual RT and PCR reaction efficiencies. Changes in gene expression of less than twofold to greater than 75-fold were readily distinguished.

Actins↗
PubMed1994

Use of the Fab fragment for immunoneutralization of somatostatin in the isolated perfused human pancreas.

The role of the somatostatin-secreting D cell in the islet remains controversial. The present study was undertaken to determine whether infusion of the Fab fragment of a highly sensitive somatostatin monoclonal antibody into the isolated, perfused human pancreas would influence insulin secretion. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs-media with 3.9 mM glucose. Sequential test periods separated by basal periods were performed with either somatostatin monoclonal antibody Fab fragment (SFab), somatostatin-14 (SS-14), or a combined infusion. Immunoneutralization of intraislet somatostatin with SFab resulted in a significant increase in both immunoreactive insulin (IRI) (1,122 +/- 497 pM) (p < 0.05) and immunoreactive C-peptide (IRC-P) secretion (146 +/- 53 pM) (p < 0.05). Infusion of SS-14 resulted in inhibition of both IRI secretion (-3,372 +/- 1,360 pM) (p < 0.05) and IRC-P secretion (-708 +/- 220 pM) (p < 0.05). Combined infusion of SFab and SS-14 reversed the inhibitory effect of exogenous SS-14 on IRI and IRC-P secretion. The data suggest that intraislet somatostatin has an inhibitory role in the regulation of B-cell secretion in the human islet and demonstrates that the Fab fragment of the somatostatin monoclonal antibody is an effective tool for immunoneutralization studies in the human pancreas. In addition, immunostaining of the donor pancreata demonstrated the presence of somatostatin-immunoreactive endocrine cells interspersed throughout the islet core and mantle. The demonstrated proximity of somatostatin-immunoreactive endocrine cells to B cells lends anatomic support to the concept that intraislet somatostatin influences insulin secretion in the human islet.

Adult↗
PubMed1994

Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects.

To determine whether induced sputum samples might provide a useful means for evaluating the effects of therapy on airway mucosal inflammation, we examined induced sputum samples obtained before and after 6 days of treatment with prednisone (0.5 mg/kg/day) or placebo in a randomized, double-blind study of 24 asthmatic subjects. Induced sputum was analyzed for total and differential cell counts and for concentrations of eosinophil cationic protein, albumin, and mucin-like glycoprotein. We found that the mean (+/- SEM) percentage of eosinophils in sputum samples from the prednisone-treated group fell from 14.1% +/- 5.0% at baseline to 1.8% +/- 0.8% after treatment, a decrease significantly greater than in the placebo-treated group (from 10.3% +/- 4.9% to 11.1% +/- 4.0%; p = 0.002). The absolute number of eosinophils also decreased significantly more in the prednisone-treated group than in the placebo-treated group (p = 0.04). In addition, eosinophil cationic protein levels in induced sputum fell more in the prednisone-treated group than in the placebo-treated group (from 324 +/- 131 ng/ml to 144 +/- 84 ng/ml vs 173 +/- 50 ng/ml to 188 +/- 47 ng/ml; p = 0.002). Furthermore, prednisone treatment was associated with a significant increase in peak expiratory flow, an effect that was significantly correlated with the decrease in eosinophil percentage in induced sputum (rs = 0.64, p = 0.04). Prednisone treatment was not associated with any significant change in the concentrations of albumin or mucin-like glycoprotein. We conclude that analysis of induced sputum is a useful noninvasive method for studying the effects of asthma therapy on airway eosinophilic inflammation.

Adult↗
PubMed1994

Prospects for reform of hospital fees in sub-Saharan Africa: a case study of Niamey National Hospital in Niger.

Hospital finance in developing countries has attracted increasing attention in recent years as economists and health planners have examined whether financial reforms will make public hospitals more financially autonomous and consequently reduce (or limit the increase in) their share of government health budgets. This paper presents estimates of the effects of some reforms of hospital user fees on total hospital revenue and on the amount of fees paid by patients in various payor categories. The reforms include special fees for non-referred patients and changes in exemptions for some categories of patients and types of care. The estimates show that doubling fees for non-referred patients increases revenue more than charging them prices equal to operating costs, because current fees are not uniformly less than operating costs. Eliminating exemptions can be as important as changing fees. For example, eliminating exemptions for surgery and inpatient diagnostic exams increases the percentage of operating costs recovered by the same amount as doubling prices for non-referred patients in estimates with an elasticity of demand equal to zero.

Fees, Medical↗
PubMed1994

Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: a method for studying allergic airway inflammation.

To determine whether analysis of the constituents of induced sputum permits detection of changes provoked by aerosolized antigen challenge, we performed sputum induction (20-minute inhalation of aerosolized 3% saline solution) before and after aerosolized allergen challenge in eight subjects with asthma. Total cell counts and cell differentials of nonsquamous cells in induced sputum samples were determined after the samples were homogenized in dithiothreitol. Centrifugation of the entire homogenized sputum sample yielded supernatant that could be analyzed for biochemical constituents. We found that the median percentage of eosinophils and neutrophils in induced sputum samples was significantly higher 4 hours after allergen challenge neutrophils in induced sputum samples was significantly higher 4 hours after allergen challenge than at baseline (12% vs 0.5%, p < 0.05; 30.5% vs 7.5%, p < 0.05) and remained high 24 hours after challenge. Median levels of eosinophil cationic protein and histamine in induced sputum supernatants were significantly higher 4 hours after challenge than at baseline (151.3 vs 39.8 ng/ml, p < 0.05; 19.4 vs 8.8 micrograms, p < 0.05) and remained significantly higher 24 hours after challenge. Tryptase was detectable in sputum from seven of the subjects, and in these subjects, we found a trend toward an increase in median tryptase levels 4 hours after allergen challenge (4.4 vs 2.2 U/L, p = 0.09). We conclude that analysis of induced sputum after aerosolized allergen challenge reveals changes in inflammatory cells and markers similar to those reported in bronchoalveolar lavage fluid and that sputum induction is a useful noninvasive method for studying allergic airway inflammation in asthma.

Administration, Inhalation↗
PubMed1994

Regulatory role of intraislet somatostatin on insulin secretion in the isolated perfused human pancreas.

This study was undertaken to determine whether intraislet somatostatin inhibits insulin secretion in the human islet. A high-affinity monoclonal somatostatin antibody was used to immunoneutralize somatostatin in the isolated, perfused human pancreas. Single pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs medium with either 3.9 or 12.9 mM glucose. Sequential test periods separated by basal periods were performed with either somatostatin-14 (SS-14), somatostatin monoclonal antibody (CURE.S6), or a combined infusion. Infusion of SS-14 resulted in inhibition of insulin secretion under both low glucose (delta X = -712 +/- 212 pM) (p < 0.05) and high glucose (delta X = -21,913 +/- 10,003 pM) (p = 0.06) conditions. Immunoneutralization of intraislet somatostatin with CURE.S6 resulted in a significant increase in insulin secretion under both low glucose (454 +/- 162 pM) (p < 0.05) and high glucose (2,177 +/- 829 pM) (p < 0.05) conditions. Combined infusion of SS-14 and CURE.S6 resulted in a reversal of the inhibitory effect of exogenous SS-14. The data suggest that intraislet somatostatin has an inhibitory role in the regulation of insulin secretion in the human islet.

Adolescent↗
PubMed1994

Parenterally or enterally administered anti-somatostatin antibody induces increased gastrin in suckling rats.

This study demonstrates the effectiveness of parenteral and oral anti-somatostatin monoclonal antibody to stimulate gastrin cell activity in suckling rats. Intraperitoneal anti-somatostatin monoclonal antibody increased serum gastrin concentration (> 2-fold), and orally administered antibodies retained their neutralizing capabilities as demonstrated by a 30% induction of gastrin synthesis. Absorption of luminal monoclonal antibody was time dependent and saturable as demonstrated by measurement of serum murine immunoglobulin G and the subsequent effects on serum gastrin and antral gastrin mRNA concentrations. Enhanced gastrin synthesis after oral administration required whole antibody in that enterally delivered F(ab')2 fragments were not absorbed and did not increase serum gastrin concentrations. In addition, pretreatment with excess Fc fragments decreased monoclonal antibody absorption and eliminated the serum gastrin response to oral monoclonal antibody. These results demonstrate that orally administered murine anti-somatostatin monoclonal antibody was rapidly and efficiently absorbed via an Fc-dependent mechanism and retained immunoneutralizing activity against endogenous somatostatin, neutralizing its inhibition of gastrin cell activity. These results indicate that orally administered monoclonal antibodies could potentially be used to determine the role of other peptides and growth factors as potential physiological regulators during the suckling period of postnatal development.

Administration, Oral↗
PubMed1994

Persistence of hormone secretion from neuroendocrine cells of aplysia after termination of electrical afterdischarge.

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

Action Potentials↗
PubMed1994

Peptide YY immunoneutralization inhibits meal-induced absorption in vivo.

BACKGROUND: Plasma peptide YY (PYY) levels rise after a meal and have recently been shown to increase small bowel-absorption. The purpose of this study was to determine whether immunoneutralization of PYY would block postprandial absorption in vivo. METHODS: Exteriorized, neurovascularly intact jejunal and ileal segments (25 cm) were created in six mongrel dogs. After a 2-week recovery luminal perfusion with an isotonic buffer, containing [14C]-polyethylene glycol as a volume marker, was used to analyze water and sodium flux after an oral meal. Each meal was accompanied by either intravenous anti-PYY (0.5 mg.kg-1.h-1) or nonspecific immunoglobulin IG (control). PYY antibody binding was determined by radioimmunoassay. RESULTS: Displacement studies showed complete PYY neutralization. In control experiments feeding increased absorption of sodium and water in both segments. PYY immunoneutralization had no effect on jejunal absorption but significantly diminished ileal absorption (p < 0.05). CONCLUSIONS: These results suggest that PYY acts selectively in the ileum to increase postprandial fluid and electrolyte absorption after a meal. Agents directed at PYY-stimulated absorption may prove to be of therapeutic benefit in patients with malabsorptive conditions.

Animals↗
PubMed1993

Antibody epitope mapping of the gastric H+/K(+)-ATPase.

Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

Amino Acid Sequence↗
PubMed1993

Endogenous somatostatin inhibits histamine release from canine gastric mucosal cells in primary culture.

The effects of somatostatin on histamine release were studied using primary cultures of canine oxyntic mucosal cells in which mast cell content was reduced by density gradient. The S6 monoclonal antibody to somatostatin, but not control antibodies, enhanced gastrin-stimulated histamine release. In the presence of S6, the somatostatin analogue SMS-201-995 (10(-7) M) inhibited gastrin-stimulated histamine release by 95%. The dose producing 50% inhibition for this inhibition was approximately 3 x 10(-10) M and was completely reversed by pertussis toxin treatment. In contrast to somatostatin, epinephrine failed to inhibit this gastrin stimulation. However, the lectin concanavalin A (ConA) also stimulated histamine release from these cultures, and this response was inhibited by epinephrine but not by somatostatin. Thus somatostatin selectively inhibited the gastrin-responsive histamine pool, which presumably is stored in oxyntic mucosal endocrine cells. In contrast, epinephrine selectively inhibits histamine release from the ConA-sensitive pool, which is presumably stored in mast cells. Furthermore, enhancement of gastrin-stimulated histamine release by immunoneutralization of somatostatin indicates an important role for endogenous somatostatin as a paracrine inhibitor of non-mast cell histamine release.

Animals↗
PubMed1993

Cytokines in metal fume fever.

Metal fume fever is a flulike illness caused by zinc oxide inhalation and accompanied by an impressive pulmonary cellular response. We hypothesized that the syndrome is mediated by cytokines released in the lung after exposure to zinc oxide fume. We carried out 26 experimental welding exposures in 23 volunteer subjects, performing postexposure bronchoalveolar lavage (BAL) 3 h (n = 6), 8 h (n = 11), or 22 h (n = 9) after exposure. We detected tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-8 (IL-8) varying in a time- and exposure-related manner. The concentration of TNF in the BAL fluid supernatant was significantly greater at 3 h than at 8 h or 22 h after exposure (p < 0.05), exhibiting a statistically significant exposure-response relationship to airborne zinc at each follow-up time period (p < 0.05). TNF concentrations were statistically correlated with those of IL-6 in BAL supernatant obtained at 22 h (r = 0.78, p = 0.01) and with concentrations of IL-8 in BAL 8 h after exposure (r = 0.85, p = 0.001). IL-6 displayed a significant exposure-response relationship to zinc (p < 0.05) at 22 h. IL-8 exhibited a significant exposure-response relationship to zinc (p < 0.05) at 8 h after exposure, a time at which IL-8 correlated with marked increases in BAL fluid polymorphonuclear leukocytes (PMN) (r = 0.7, p = 0.01). Although we also detected interleukin-1 (IL-1) in BAL samples, this cytokine did not demonstrate a statistically significant exposure response. TNF, IL-6, and IL-8 in BAL fluid supernatant concentrations increased in a time and exposure-dependent fashion after zinc oxide welding fume exposure. The time course of increased cytokines, their correlations with one another and with PMN in the BAL fluid, and the consistency of our findings with the known kinetics and actions of these cytokines support the hypothesis that a network of cytokines is involved in the pathogenesis of metal fume fever.

Adult↗
PubMed1993

Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects.

To determine the feasibility of cellular and biochemical analysis of sputum induced after inhalation of hypertonic (3%) saline, we analyzed sputum induced in 10 healthy and in 18 asthmatic subjects. We also analyzed saliva samples from all subjects. The entire sputum sample and the saliva sample were reduced using dithiothreitol, and cell counts and differentials were determined. Biochemical analysis was performed on sputum and saliva supernatants obtained after centrifugation. We found that induced sputum from asthmatic subjects had a higher percentage of eosinophils [8.1 +/- 3.43 (mean +/- SEM) versus 0.03 +/- 0.02%, p < 0.009] (after excluding squamous cells) and also had higher levels of albumin (232.3 +/- 54.8 versus 79.5 +/- 9.7 micrograms/ml, p < 0.02), fibrinogen (44.2 +/- 11.6 versus 11.9 +/- 2.5 micrograms/ml, p < 0.008) and eosinophil cationic protein (ECP) (142.6 +/- 34.2 versus 26.1 +/- 4.7 ng/ml, p < 0.006) but not of histamine or tryptase. In saliva, squamous cells made up more than 99% of the cells in both groups, and protein concentrations were not significantly different. We conclude that cellular and biochemical analysis of induced sputum is feasible in healthy and in asthmatic subjects and that it reveals differences similar to those reported from analyses of bronchial lavage fluid.

Adult↗
PubMed1992

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.

Amino Acid Sequence↗