ABSTRACT
The central role of human pancreatic glucokinase in insulin secretion and, consequently, in maintenance of blood glucose levels has prompted investigation into identification of ATP-binding site residues and examination of ATP- and glucose-binding interactions. Because glucokinase has been resistant to crystallization, computer generated homology models were developed based on the X-ray crystal structure of the COOH-terminal domain of human brain hexokinase 1 bound to glucose and ADP or glucose and glucose-6-phosphate. Human pancreatic glucokinase mutants were designed based upon these models and on ATPase domain sequence conservation to identify and characterize potential glucose and ATP-binding sites. Specifically, mutants Asp78Ala, Thr82Ala, Lys90Ala, Lys102Ala, Gly227Ala, Thr228Ala, Ser336Leu, Ser411Ala, and Ser411Leu were constructed, expressed, purified, and kinetically characterized under steady-state conditions. Compared to their respective wild type controls, several mutants demonstrated dramatic changes in V(max), cooperativity of glucose binding and S(0.5) for ATP and glucose. Results suggest a role for Asp78, Thr82, Gly227, Thr228, and Ser336 in ATP binding and indicate these residues are essential for glucose phosphorylation by human pancreatic glucokinase.
Subject(s)
Adenosine Triphosphate/metabolism , Glucokinase/metabolism , Pancreas/enzymology , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Computer Simulation , Crystallography, X-Ray , Glucokinase/chemistry , Glucokinase/genetics , Glucose/metabolism , Glucose-6-Phosphate/metabolism , Humans , Kinetics , Molecular Sequence Data , Mutation , Phosphorylation , Substrate SpecificityABSTRACT
A direct flow injection analysis/mass spectrometry (FIA/MS) method was developed for the quantification of polyethylene glycol. The method was used for the evaluation of distribution uniformity and mixing homogeneity of polyethylene glycol 300 (PEG 300) as a component in drug formulation mixtures. In the method, five of the most intense ions of the PEG 300 oligomer were chosen for selected ion monitoring (SIM) by mass spectrometry. Standard calibration curves were established, using either single channel SIM or the summed intensity of all five SIM channels plotting against the standard concentrations. Both calibration approaches produced comparable results on quantification. The feasibility of the method was demonstrated using both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The method provided fast and sensitive quantification of PEG 300 without tedious chromatographic separation or sample preparation. The method has been successfully adopted for the evaluation of the mixing process in drug formulations.
Subject(s)
Polyethylene Glycols/analysis , Calibration , Chemistry, Pharmaceutical , Excipients , Flow Injection Analysis , Indicators and Reagents , Mass Spectrometry , Quality Control , Spectrometry, Mass, Electrospray IonizationABSTRACT
OBJECTIVES/HYPOTHESIS: Lipomas of the retropharyngeal space are rare and do not cause symptoms until they reach a large size. Although retropharyngeal lipoma is an uncommon entity, several reports of it appear in the literature, and the treatment has routinely been surgical excision. Such fatty tumors also carry the rare possibility of being liposarcomas, which further warrants their excision. We present the case of a lipoma of the retropharyngeal space extending from the nasopharynx to the superior mediastinum causing symptoms of obstructive sleep apnea. The patient had multiple medical problems and was on a regimen of anticoagulation therapy; therefore, he opted against surgical treatment. He has used continuous positive airway pressure and has been followed clinically and radiographically for 5 years. Radiographic follow-up of a retropharyngeal lipoma after a needle biopsy confirming its benign nature is a legitimate means of management of this rare condition. STUDY DESIGN: Case report of a 64-year-old man presenting with this rare lesion. METHODS: Computed tomography-guided needle biopsy of the mass was performed to obtain tissue diagnosis. Thereafter, the patient has been followed for 5 years with annual magnetic resonance imaging scans to determine growth or changes of the retropharyngeal mass. RESULTS: Fine-needle aspiration of the mass revealed mature adipose tissue intermixed with fibroconnective tissue, consistent with lipoma. Based on this result, we opted to follow this patient with serial magnetic resonance imaging scans, which have shown no change in size. There has also been no change in the patient's symptoms. CONCLUSIONS: Large lipomas warrant excision, especially when their location produces pressure symptoms. However, when surgical morbidity is high, fine-needle aspiration biopsy and serial magnetic resonance imaging scans are a safe alternative.
