ABSTRACT
BACKGROUND: Pulmonary complications cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant (AHSCT). Bronchoscopy with targeted bronchoalveolar lavage (BAL) is often used in AHSCT patients with suspected lower respiratory tract infection (LRTI) to help guide management. AIM: To evaluate how positive BAL results change antimicrobial management of AHSCT recipients with suspected LRTI. METHODS: We performed a retrospective review of BAL results from January 2014 to July 2016 for 54 AHSCT recipients. A positive BAL was determined by culture, multiplex polymerase chain reaction (PCR), Aspergillus galactomannan antigen (AGA), and cytology. FINDINGS: BAL was positive for infectious etiologies in 63%, and antimicrobials were adjusted in 48/54 (89%) of patients. Antibacterial escalation was predicted by a positive BAL bacterial culture (OR 7.61, P=0.017). Antibiotic de-escalation was more likely with an elevated AGA (OR 3.86, P=0.035). Antiviral initiation was more likely with positive BAL multiplex PCR (OR 17.33, P=0.010). Antifungals were more likely to be escalated or changed with an elevated AGA (OR 4.33, P=0.020). The patients with a negative BAL were more likely to be started on steroids (OR 0.19, P= 0.043). CONCLUSIONS: BAL was helpful to determine the etiology of pulmonary complications and optimize antimicrobials. The addition of AGA and multiplex PCR to standard BAL significantly impacted de-escalating antibiotics and adjusting antifungals to provide adequate coverage. The association with an elevated AGA with antibacterial de-escalation highlights a new role for BAL in antimicrobial optimization.
ABSTRACT
BACKGROUND: Chlamydia trachomatis (Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. Ct strain-specific differences in clinical trachoma suggest that genetic polymorphisms in Ct may contribute to the observed variability in severity of clinical disease. METHODS: Using Ct whole genome sequences obtained directly from conjunctival swabs, we studied Ct genomic diversity and associations between Ct genetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea-Bissau, West Africa. RESULTS: All Ct sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion in trpA resulting in a truncated non-functional protein and the ocular tyrosine repeat regions present in tarP associated with ocular tissue localization. We have identified 21 Ct non-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs within pmpD (odds ratio, OR = 4.07, p* = 0.001) and tarP (OR = 0.34, p* = 0.009). Eight synonymous SNPs associated with disease severity were found in yjfH (rlmB) (OR = 0.13, p* = 0.037), CTA0273 (OR = 0.12, p* = 0.027), trmD (OR = 0.12, p* = 0.032), CTA0744 (OR = 0.12, p* = 0.041), glgA (OR = 0.10, p* = 0.026), alaS (OR = 0.10, p* = 0.032), pmpE (OR = 0.08, p* = 0.001) and the intergenic region CTA0744-CTA0745 (OR = 0.13, p* = 0.043). CONCLUSIONS: This study demonstrates the extent of genomic diversity within a naturally circulating population of ocular Ct and is the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocular Ct pathogenesis and disease transmission.
Subject(s)
Chlamydia trachomatis/genetics , Genome, Bacterial , Severity of Illness Index , Trachoma/microbiology , Conjunctiva/pathology , Endemic Diseases , Genetic Markers , Guinea-Bissau , Humans , Likelihood Functions , Phenotype , Phylogeny , Polymorphism, Single Nucleotide/genetics , Trachoma/pathology , Whole Genome SequencingABSTRACT
Rabbit anti-thymocyte globulin (ATG) is used as prophylaxis against GVHD following allogeneic hematopoietic cell transplantation (HCT). At our institution, ATG is exclusively used in the conditioning of matched unrelated donor (URD) transplant recipients. A total of 50 URD HCT recipients who received ATG (ATG group) were retrospectively compared with 48 matched related donor (MRD) HCT recipients who did not receive ATG (no ATG group). There were no significant differences between the groups in rates of day 100 mortality, acute GVHD or relapse. Chronic GVHD incidence was significantly lower in the ATG group (P = 0.007). At a median follow-up of 36 months in the entire cohort, 50% patients are alive in the ATG group and 63% of the patients are alive in the no ATG group (P = 0.13). We conclude that the administration of ATG to patients undergoing URD HCT preserves the anti-leukemia benefit of the transplant, while reducing the risk of developing GVHD, resulting in OS rates that are comparable to MRD HCT recipients.
Subject(s)
Antilymphocyte Serum/therapeutic use , Hematopoietic Stem Cell Transplantation/methods , Transplantation Conditioning/methods , Cohort Studies , Female , Graft vs Host Disease/prevention & control , Humans , Male , Middle Aged , Myelodysplastic Syndromes/surgery , Precursor Cell Lymphoblastic Leukemia-Lymphoma/surgery , Randomized Controlled Trials as Topic , Retrospective Studies , Survival Analysis , Tissue Donors , Transplantation, Homologous , Treatment Outcome , Unrelated DonorsABSTRACT
BACKGROUND: Leprosy is complicated by immunological reactions which can occur before, during and after successful completion of multidrug therapy. Genetic studies have suggested that polymorphisms in toll-like receptors (TLRs) may affect the susceptibility of an individual with leprosy to developing Type 1 reactions. OBJECTIVES: To examine the gene and protein expression of TLRs in the cutaneous lesions of leprosy Type 1 reactions at the onset of reaction and during systemic corticosteroid therapy. METHODS: Patients who were being treated for leprosy type 1 reactions with corticosteroids as part of a randomized controlled trial of corticosteroid treatment had skin biopsies performed before, during and at the end of treatment. The gene and protein expression of TLR2 and TLR4 were measured. RESULTS: We have demonstrated that the gene hARP-P0 is a suitable control gene for TLR gene expression studies in this population. The gene and protein expression of TLR2 and TLR4 were both reduced significantly during corticosteroid treatment. CONCLUSIONS: This is the first study to examine the expression of TLR2 and TLR4 in vivo in individuals experiencing leprosy Type 1 reactions. The data support the possibility of an important role for TLR2 and TLR4 in the pathogenesis of this important complication of leprosy.
Subject(s)
Glucocorticoids/therapeutic use , Leprosy/drug therapy , Toll-Like Receptor 2/physiology , Toll-Like Receptor 4/physiology , Adolescent , Adult , Analysis of Variance , Antibiotics, Antitubercular/therapeutic use , DNA, Complementary/biosynthesis , Drug Therapy, Combination , Female , Gene Expression , Humans , Immunohistochemistry , Leprosy/genetics , Leprosy/mortality , Male , Methylprednisolone/therapeutic use , Middle Aged , Prednisolone/therapeutic use , Toll-Like Receptor 2/drug effects , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/drug effects , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Young AdultABSTRACT
BACKGROUND: Treatment with fluoroquinolones is associated with the development of Clostridium difficile and extended spectrum beta-lactamase-producing bacteria (ESBL). Clostridium difficile and ESBL are resistant to many antibiotics and each may cause pouchitis after restorative proctocolectomy (RPC) refractory to empirical antibiotic therapy. AIM: To assess the prevalence and establish risk factors for the development of ESBL and Clostridium difficile toxins (CDT) in RPC patients with recurrent or refractory pouchitis under follow-up at our institution over a 1-year period. METHOD: An enzyme-linked immunosorbent assay was used to detect CDT and a culture technique was used to identity ESBL in faecal samples. All patients had previously received fluoroquinolone treatment. RESULTS: Forty-eight patients (35 (74%) men; median age 42 years) underwent testing at a median interval from RPC of 8 (range 1-25) years. No patient had a positive CDT result, but ESBL bacteria were identified in 16 (33%) samples. ESBL positivity was significantly related to prepouch ileitis (P = 0.035) and maintenance antibiotic therapy (P = 0.039). CONCLUSIONS: Extended spectrum beta-lactamase, but not CDT, is a common finding in faecal samples from patients with recurrent or refractory pouchitis. Treatment with maintenance antibiotics and prepouch ileitis are risk factors for developing ESBL-producing bacteria.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Clostridium Infections/microbiology , Enterobacteriaceae Infections/microbiology , Pouchitis/microbiology , Proctocolectomy, Restorative/adverse effects , beta-Lactamases/metabolism , Adult , Clostridioides difficile/isolation & purification , Drug Resistance , Enterobacteriaceae/isolation & purification , Female , Follow-Up Studies , Humans , Male , Microbial Sensitivity Tests , Middle Aged , Risk Factors , Young AdultABSTRACT
Controlled studies that address risk factors for, and clinical outcomes after, infection with extended-spectrum beta-lactamase (ESBL)-producing organisms are scant, particularly in the intensive care unit (ICU). Our objectives were to elucidate risk factors for the acquisition of ESBL-producing organisms in ICU; and to compare mortality in patients with ESBL- and non-ESBL bloodstream infections (BSIs) after controlling for disease severity and timeliness of appropriate antibiotic therapy. A retrospective cohort study was undertaken in the ICU from March 2004 to May 2006. Cases included all adult ICU patients with a BSI due to an ESBL-producing E. coli or Klebsiella spp. (N=16); controls (N=39) comprised ICU patients with a BSI caused by a non-ESBL-producing E. coli or Klebsiella spp. Disease severity was measured using APACHE (Acute Physiological Assessment and Chronic Health Evaluation) and SOFA (Sequential Organ Failure Assessment) scores. Outcomes were recorded as discharge or death due to all causes. Although no statistically significant associations were demonstrated between individual risk factors and the acquisition of an ESBL-producing organism, appropriate therapy was delayed in cases (OR: 9.17; 95% CI: 2.00-42.20; P=0.0005) and survival estimates demonstrated a significantly increased early (<25 days after infection) mortality (OR for death 3.93; 95% CI: 1.05-14.63; P=0.03). Mortality in ICU, when adjusted for disease severity and appropriate antimicrobial therapy, though significant needs to be treated with caution due to the small number of cases (N=16 in 2 years). We believe that a high index of suspicion, early appropriate therapy and strict adherence to infection control are indicated in all patients at risk in ICU.
Subject(s)
Bacteremia/mortality , Cross Infection/mortality , Escherichia coli Infections/mortality , Klebsiella Infections/mortality , Adolescent , Adult , Aged , Anti-Bacterial Agents/therapeutic use , Bacteremia/drug therapy , Bacteremia/microbiology , Child , Child, Preschool , Cross Infection/drug therapy , Cross Infection/microbiology , Drug Resistance, Multiple, Bacterial , Escherichia coli/enzymology , Escherichia coli/isolation & purification , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Female , Humans , Infant , Infant, Newborn , Intensive Care Units , Klebsiella/enzymology , Klebsiella/isolation & purification , Klebsiella Infections/drug therapy , Klebsiella Infections/microbiology , London/epidemiology , Male , Middle Aged , Proportional Hazards Models , Retrospective Studies , Risk Factors , Severity of Illness Index , Treatment Outcome , beta-Lactamases/biosynthesisABSTRACT
DNA enrichment by allele-specific hybridization (DEASH) was used as a means to isolate individual alleles of the killer cell immunoglobulin-like receptor (KIR2DL4) gene from heterozygous genomic DNA. Using long-template polymerase chain reaction (LT-PCR), the complete KIR2DL4 gene was amplified from a cell line that had previously been characterized for its KIR gene content by PCR using sequence-specific primers (PCR-SSP). The whole gene amplicons were sequenced and we identified two heterozygous positions in accordance with the predictions of the PCR-SSP. The amplicons were then hybridized to allele-specific, biotinylated oligonucleotide probes and through binding to streptavidin-coated beads, the targeted alleles were enriched. A second PCR amplified only the exonic regions of the enriched allele, and these were then sequenced in full. We show DEASH to be capable of enriching single alleles from a heterozygous PCR product, and through sequencing the enriched DNA, we are able to produce complete coding sequences of the KIR2DL4 alleles in accordance with the typing predicted by PCR-SSP.
Subject(s)
DNA/genetics , DNA/isolation & purification , Receptors, Immunologic/genetics , Alleles , Base Sequence , Humans , Magnetics , Nucleic Acid Hybridization/methods , Oligonucleotide Probes/genetics , Polymerase Chain Reaction/methods , Receptors, KIR , Receptors, KIR2DL4ABSTRACT
OBJECTIVE: To assess the safety of benzyl benzoate lotion (BBL) and permethrin, topical treatments for scabies, during pregnancy. DESIGN: A retrospective controlled cohort study. POPULATION: Refugee and migrant women attending antenatal clinics (ANC) on the Thai-Burmese border between August 1993 and April 2006. METHODS: Women treated with either BBL (25%) or permethrin (4%) were identified from a manual search of antenatal records. Each case of scabies was matched with four scabies-free controls for gravidity, age, smoking status, malaria, period of treatment and gestational age at treatment. Conditional Poisson regression was used to estimate risk ratios for outcomes of pregnancy (proportion of abortions, congenital abnormalities, neonatal deaths, stillbirths and premature babies), mean birthweight and estimated median gestational age, for scabies and scabies-free women, independently for BBL and permethrin. RESULTS: There were no statistically significant differences in pregnancy outcomes between women who were treated with either BBL (n = 444) compared with their matched controls (n = 1,776) or permethrin (n = 196) treated women and their matched controls (n = 784). Overall, only 10.9% (n = 66) of treatments were in the first trimester. Retreatment rates were higher with BBL 16.4%, than permethrin 9.7%, P = 0.038. Scabies was more common during cooler periods. CONCLUSION: We found no evidence of adverse effects on pregnancy outcome due to topical 25% BBL or 4% permethrin.
Subject(s)
Benzoates/adverse effects , Insecticides/adverse effects , Permethrin/adverse effects , Pregnancy Complications, Parasitic/prevention & control , Scabies/prevention & control , Administration, Topical , Benzoates/administration & dosage , Case-Control Studies , Cohort Studies , Female , Humans , Insecticides/administration & dosage , Permethrin/administration & dosage , Pregnancy , Pregnancy Outcome , Refugees , Retrospective StudiesABSTRACT
Killer-cell immunoglobulin-like receptor (KIR) genes are highly polymorphic and polymorphisms have been found in all KIR exons. Although less is known of the introns, these also appear to be polymorphic. To generate a comprehensive database of KIR genomic sequences, which will aid in the design of KIR typing reagents, we have established a method for cloning and sequencing of KIR genes from genomic DNA. We cloned and sequenced the entire KIR2DL4 gene from genomic DNA using long template touchdown PCR and high capacity cloning vectors. Overlapping secondary amplicons were modified to include a nucleotide analogue that reduced sequencing problems associated with secondary structure formation in the KIR sequence. Using a modified sequencing chemistry we were able to sequence approximately 11,000 bases confirming the previously published KIR2DL4*005 allele sequence.
Subject(s)
Alleles , DNA/genetics , Genome, Human , Receptors, Immunologic/genetics , Sequence Analysis, DNA , Cloning, Molecular , DNA Primers , Humans , Killer Cells, Natural , Molecular Sequence Data , Polymerase Chain Reaction , Receptors, KIR , Receptors, KIR2DL4ABSTRACT
OBJECTIVES: To determine the cost-effectiveness of managing an abnormal Papanicolaou (PAP) smear during pregnancy with a single colposcopic exam and biopsies, followed by Pap smears in each subsequent trimester of pregnancy and 8 weeks postpartum. MATERIALS AND METHODS: We reviewed 84 pregnant women with cervical intraepithelial neoplasia (CIN) between 1983 and 1991, testing the accuracy of an initial biopsy and subsequent Pap smears, to follow the progression (or regression) of disease as determined by postpartum biopsy or Pap smears. RESULTS: In 26 women with CIN1, 2 (8%) progressed to CIN3. In 29 women with CIN2, 5 (17%) progressed to CIN3. Of 29 patients with CIN3, 20 (69%) remained at CIN3 and 2 (6%) progressed to microinvasive carcinoma postpartum, confirmed by conization. No invasive carcinoma was missed. The cost of colposcopy with biopsies and Pap smear is $304, whereas cost of a Pap smear only is $30. CONCLUSIONS: Single colposcopy with biopsies at the beginning of pregnancy and Pap smears during subsequent trimesters and postpartum should be adequate follow-up to prevent progression to invasive cancer and represents a significant cost savings.
ABSTRACT
Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.
Subject(s)
Alkaline Phosphatase/metabolism , Escherichia coli/enzymology , Peptide Hydrolases/metabolism , Amino Acid Sequence , Circular Dichroism , Drug Stability , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Kinetics , Macromolecular Substances , Molecular Sequence Data , Peptide Fragments/metabolism , Phosphates/metabolism , Protein Conformation , Serine Endopeptidases/metabolism , Structure-Activity Relationship , Trypsin/metabolismABSTRACT
Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, we examined the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial [3H]tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of [3H]tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.
Subject(s)
Hemorrhage/blood , Hypotension/blood , Plasma/metabolism , Spinal Cord/metabolism , Tyrosine/metabolism , Amino Acids/blood , Animals , Catecholamines/biosynthesis , Catecholamines/metabolism , Male , Rats , Rats, Sprague-Dawley , Tyrosine/bloodABSTRACT
The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.
Subject(s)
Alkaline Phosphatase/metabolism , Apoenzymes/pharmacology , Apoproteins/pharmacology , Chlorides , Escherichia coli/enzymology , Trypsin/pharmacology , Zinc Compounds , Calorimetry, Differential Scanning , Kinetics , Macromolecular Substances , Magnesium/pharmacology , Magnesium Chloride , Molecular Weight , Zinc/pharmacologyABSTRACT
Administration of the artificial sweetener aspartame (L-aspartylphenylalanylmethyl ester; 200 mg/kg) by gavage to rats caused large increments in brain and plasma levels of phenylalanine and its product tyrosine. Glucose administration (3 g/kg, by gavage, a dose sufficient to cause insulin-mediated reductions in plasma levels of the large neutral amino acids leucine, isoleucine, and valine) also elevated brain phenylalanine and tyrosine, and enhanced the increments caused by the aspartame, nearly doubling the rise in brain phenylalanine. Each animal's brain phenylalanine or tyrosine levels were highly correlated (r = 0.97 and 0.99, respectively) with its plasma phenylalanine or tyrosine ratios, affirming that aspartame's effects on the brain amino acids result from the changes it produces in plasma composition. As described previously, glucose consumption increased brain tryptophan levels, and consequently, brain levels of the 5-hydroxyindoles serotonin and 5-hydroxyindoleacetic acid. Aspartame alone had no effect on these compounds but completely blocked the changes in 5-hydroxyindoles caused by glucose. Each animal's brain level of tryptophan (r = 0.89) and 5-hydroxyindoles (r = 0.74) was also significantly correlated with its plasma tryptophan ratio, affirming that the effects of glucose or aspartame on these brain constituents also result from the changes they produce in plasma composition. The aspartame-glucose combination also reduced brain levels of leucine, isoleucine, and valine to a significantly greater extent than aspartame or glucose alone. These observations indicate that high aspartame doses can generate major neurochemical changes in rats, especially when consumed along with carbohydrate-containing foods. However, they should not in any way be interpreted as demonstrating that aspartame significantly affects the human brain.
Subject(s)
Amino Acids/metabolism , Aspartame/pharmacology , Brain/drug effects , Dipeptides/pharmacology , Glucose/pharmacology , Amino Acids/blood , Animals , Brain/metabolism , Drug Interactions , Hydroxyindoleacetic Acid/analysis , Male , Phenylalanine/analysis , Rats , Rats, Inbred Strains , Serotonin/analysis , Tyrosine/analysisABSTRACT
Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.
Subject(s)
Alkaline Phosphatase/metabolism , Cadmium/metabolism , Calorimetry, Differential Scanning , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Kinetics , Phosphates/pharmacology , Protein BindingABSTRACT
A trypsin-modified form of Escherichia coli alkaline phosphatase has been isolated, purified, and characterized. The native enzyme, previously thought to be resistant to proteases, shows a loss of 20% of its activity after a 30-min exposure to 10% trypsin. No further loss is seen after 3 h; this is in contrast to the apoenzyme which loses essentially all restorable activity (addition of saturating Zn(II) and Mg(II) restores activity to the apoenzyme) when exposed to trypsin. Under these conditions, a single major peptide is produced, cleaved at the Arg-10 Ala-11 bond, which is purified using a chromatographic technique that separates proteins according to their pI (chromatofocusing). This modified alkaline phosphatase has a Vmax of 2000 mumol/h/mg (1 M Tris, pH 8.0, 20 degrees C, 1 mM p-nitrophenolphosphate) which is 22% less than the Vmax for the native enzyme. The Km for p-nitrophenolphosphate is lower for trypsin-modified alkaline phosphatase than for the native enzyme, 1.9 X 10(-5) and 4 X 10(-5) M, respectively. The KI for Pi for the native enzyme is 1.5 X 10(-5) M and for trypsin-modified alkaline phosphatase is 1 X 10(-5) M, suggesting that the reduction in Vmax is due to a reduction in the rate constant for Pi dissociation. Differential scanning calorimetry results indicate differences in the stabilities of the two species. The trypsin-modified alkaline phosphatase has a Tm of 90 degrees C which is lower than that for the reconstituted apoenzyme (93.5 degrees C) or for the native enzyme (98.5 degrees C). This modified form of alkaline phosphatase may prove to be valuable in studies concerning subunit interactions in this system as the deleted decapeptide occurs at the subunit interface region in the native structure.