Kenneth A. Skau

Acetylcholinesterase molecular forms in serum and erythrocytes of laboratory animals

The distribution of the molecular forms of acetylcholinesterase in the blood of various animal species was examined. The globular tetrameric form was most frequently observed in serum but mouse serum also contained a globular monomer. Globular monomers (rat) dimers (mouse, dog, rabbit) and tetramers (dog) were found in erythrocytes. Interspecies differences make it difficult to formulate a cohesive theory as to the origin and function of blood-borne enzymes.

Reduced Renal Accumulation and Toxicity of Cisplatin in Experimental Galactosemia

Abstract The kidneys of streptozotocin (STZ)-diabetic rats are resistant to certain toxic effects of the antineoplastic drug cisplatin. The mechanism is unknown. This study used the galactosemic rat model to test the hypothesis that the apparent diabetes-induced protection is due to changes in the kidney secondary to chronically elevated hexose concentrations. Galactosemic rats are normoinsulinemic and are free from many of the multiple biochemical abnormalities seen in STZ diabetics. The experiments compared renal cortical platinum (Pt) and blood urea nitrogen (BUN) levels after intraperitoneal injection of 5 mg/kg of cisplatin in galactosemic, STZ-diabetic, and age-matched nondiabetic Sprague-Dawley rats. Nephrotoxicity was defined as a BUN concentration ratio (after to before cisplatin) >2.5. The results demonstrate that the kidneys of both galactosemic and STZ-diabetic rats became resistant to cisplatin-induced elevation of BUN and, further, that the development of the protection was related to the duration of the diabetic state. Although the protective effect developed more slowly in the galactosemic rats, the attenuation of the rise in BUN was ultimately comparable to that seen in STZ diabetics. Renal cortex [Pt] after cisplatin injection was significantly lower in galactosemics and STZ diabetics compared with age-matched nondiabetics, with the order nondiabetics > galactosemics > STZ diabetics. It was noted, however, that renal Pt accumulation was maximally depressed within 4 weeks of experimental diabetes, whereas the BUN ratio continued to decline with increasing duration of both galactosemia and STZ diabetes. Thus, reduced renal Pt accumulation cannot by itself explain the progressive attenuation of the toxicity. The results support the hypothesis and suggest that the galactosemic rat will be a useful model for mechanistic study of diabetes-induced protection from cisplatin nephrotoxicity.

Characterization of Acetylcholinesterase in Caco-2 Cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

Phenylmethylsulfonyl fluoride inhibitory effects on acetylcholinesterase of brain and muscle.

Differential inhibition of brain versus peripheral acetylcholinesterase (AChE) by phenylmethylsulfonyl fluoride (PMSF) suggested that PMSF might preferentially inhibit different AChE molecular forms. AChE inhibition was examined after systemic and in vitro PMSF treatment. Systemic administration resulted in no overt behavioral changes but produced a 71% reduction in brain AChE; hemidiaphragm, extensor digitorum longus and soleus muscles showed 65, 50 and 41% reductions. Muscle asymmetric AChE was reduced to the greatest extent (50-80%). The tetrameric form was inhibited in brain and hemidiaphragm (60-76%) but spared in other muscles (18-22%). Monomeric AChE was spared in all tissues. When PMSF was added to a muscle homogenate all forms were inhibited equally. Purified monomer and tetramer forms were inhibited equally in vitro. These results suggest that PMSF inhibition of AChE is a consequence of a selective inhibition of membrane-associated forms and that the apparent brain selectivity is related to the greater fraction of membrane-associated AChE in brain.

Characteristics of the myocardial PM-FABP: Effect of diabetes mellitus

Properties of the myocardial PM-FABP were studied in normal and STZ-diabetic rats. The fluorescent fatty acids trans-parinaric and cis-parinaric acids were used as analogs of straight-chain (saturated) and kinked-chain (unsaturated) fatty acids respectively. Parinaric acid binding was sensitive to trypsin. Trans-parinaric acid binding was more sensitive to this protease than the binding of cis-parinaric acid. Based on the difference in sensitivity of parinaric acid binding we believe that there are two separate binding sites associated with myocardial PM-FABP; one for unsaturated fats and the other for saturated fats. Diabetes enhanced both cis- and trans-parinaric acid binding capacity in cardiomyocytes; cis-parinaric acid by 2 fold and trans-parinaric acid by 2.6 fold. In addition, there was a concomitant accumulation of free fatty acids and triglycerides in the hearts of the diabetic animals. There was a 2.2 fold increase for fatty acids and a 1.6 fold increase for trigylcerides. This association between myocardial fatty acid build-up and enhanced myocardial PM-FABP during diabetes suggest that this carrier protein might have contributed to lipid accumulation in the hearts of the diabetic rats.

Exploring the Hexokinase Glucose Binding Site Through Correlation Analysis and Molecular Modeling of Glucosamine Inhibitors

A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase. All derivatives exhibited competitive inhibition kinetics with respect to glucose. Quantitative structure-activity relationships were derived from the resulting inhibition data. The most significant equation demonstrated the existence of highly specific steric effects for the seven meta-substituted benzoylglucosamines included in the relationship. Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents.

EFFECT OF HYDRALAZINE ON MYOCARDIAL PLASMA MEMBRANE FATTY ACID BINDING PROTEIN (PM-FABP) DURING DIABETES MELLITUS

Chronic treatment with the antihypertensive drug hydralazine did not affect the hyperglycemic state of streptozotocin (STZ)-diabetic rats but did prevent the serum hyperlipidemia that is synonymous with these diabetic animals. After 6 weeks, untreated STZ-diabetic rats exhibited a 659% increase in serum triglycerides and 292% increase in serum cholesterol versus age-matched non-diabetic rats. Hydralazine-treated STZ-diabetic rats had serum triglyceride and cholesterol levels that did not differ from controls. Myocytes from control rats showed a preference for binding of the unsaturated fatty acid analog cis-parinaric acid vs the saturated fatty acid analog trans-parinaric acid. This preference was not altered in STZ-diabetic rat myocytes; hydralazine-treatment of STZ-diabetic rats also showed no change in fatty acid preference. STZ-diabetes caused a decrease in the affinity (Kd) for the trans, but not the cis-parinaric acid. However, total binding of both analogs was increased in STZ-diabetes. Hydralazine treatment of STZ-diabetic rats resulted in even greater total binding of both analogs. Affinity for the trans analog was further decreased in these hydralazine-treated rats, but the affinity for the cis analog was increased beyond control animals. These results suggest that the diabetic state influences the binding characteristics of the myocardial PM-FABP and that hydralazine, while reducing serum lipids in diabetes, has complex effects on the fatty acid binding by this protein.

Distribution and solubilization of the molecular forms of acetylcholinesterase in rat urinary bladder and sphincter

The distribution and solubility of the molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) were examined in adult, male rat bladder body and sphincter. Four distinct forms of the enzyme solubilized from bladder body were separated on sucrose density gradients. Two of the forms (A8 and A12) corresponded to asymmetric proteins and were solubilized with high ionic strength buffer. The other two forms represented globular forms. The smallest form (G1) was soluble in low ionic strength buffer without detergent. About 33% of the larger globular form (G4) required detergent for solubilization. There were only minor differences in the distribution of these forms in juvenile rat bladder and adult, female rat bladders. Sphincter tissue lacked one of the asymmetric forms but otherwise resembled the bladder bodies. These results demonstrate that some smooth muscle organs have significant amounts of asymmetric, as well as globular, forms of acetylcholinesterase and suggest that additional studies should be performed to characterize the enzyme in this and other smooth muscle systems.

Final Report of the 2006-2007 Council of Faculties.

I would like to take this opportunity to thank all officers of COF and committee chairs and members who contributed to a successful year.