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1.
J Comput Aided Mol Des ; 10(3): 213-32, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8808738

ABSTRACT

The design of enzyme mimics with therapeutic and industrial applications has interested both experimental and computational chemists for several decades. Recent advances in the computational methodology of restrained molecular dynamics, used in conjunction with data obtained from two-dimensional 1H NMR spectroscopy, make it a promising method to study peptide and protein structure and function. Several issues, however, need to be addressed in order to assess the validity of this method for its explanatory and predictive value. Among the issues addressed in this study are: the accuracy and generizability of the GROMOS peptide molecular mechanics force field; the effect of inclusion of solvent on the simulations; and the effect of different types of restraining algorithms on the computational results. The decapeptide Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, which corresponds to the sequence of ACTH1-10, has been synthesized, cyclized, and studied by two-dimensional 1H NMR spectroscopy. Restrained molecular dynamics (RMD) and time-averaged restrained molecular dynamics (TARMD) simulations were carried out on four different distance-geometry starting structures in order to determine and contrast the behavior of cyclic ACTH1-10 in vacuum and in solution. For the RMD simulations, the structures did not fit the NOE data well, even at high values of the restraining potential. The TARMD simulation method, however, was able to give structures that fit the NOE data at high values of the restraining potential. In both cases, inclusion of explicit solvent molecules in the simulation had little effect on the quality of the fit, although it was found to dampen the motion of the cyclic peptide. For both simulation techniques, the number and size of the NOE violations increased as the restraining potential approached zero. This is due, presumably, to inadequacies in the force field. Additional TARMD vacuum-phase simulations, run with a larger memory length or with a larger sampling size (16 additional distance-geometry structures), yielded no significantly different results. The computed data were then analyzed to help explain the sparse NOE data and poor chymotryptic activity of the cyclic peptide. Cyclic ACTH1-10, which contains the functional moieties of the catalytic triad of chymotrypsin, was evaluated as a potential mimic of chymotrypsin by measurement of the rate of hydrolysis of esters of L- and D-phenylalanine. The poor rate of hydrolysis is attributed to the flexibility of the decapeptide, the motion of the side chains, which result in the absence of long-range NOEs, the small size of the macrocycle relative to that of the substrate, and the inappropriate orientation of the Gly, His, and Ser residues. The results demonstrate the utility of this method in computer-aided molecular design of cyclic peptides and suggest structural modifications for future work based on a larger and more rigid peptide framework.


Subject(s)
Adrenocorticotropic Hormone/chemical synthesis , Peptide Fragments/chemical synthesis , Peptides, Cyclic/chemical synthesis , Adrenocorticotropic Hormone/chemistry , Amino Acid Sequence , Catalysis , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide Fragments/chemistry , Peptides, Cyclic/chemistry , Protein Structure, Tertiary , Serine Endopeptidases/chemistry , Solvents
2.
Biochim Biophys Acta ; 1204(1): 124-8, 1994 Jan 11.
Article in English | MEDLINE | ID: mdl-8305469

ABSTRACT

Fourier transform infrared spectroscopy was used to examine the effect of calcium binding on the secondary structure of two inhibited bovine beta-trypsins. Neither the diisopropyl fluorophosphate- nor benzamidine-inhibited forms showed detectable secondary structure perturbation upon calcium binding at pD 6.9 and 5.0, respectively. Considered in light of the recent assignment of an amide I' band to the autolysis loop of bovine beta-trypsin, these results contradict the generally held hypothesis that calcium slows trypsin autolysis by induction of a conformational change at this site and support the recent contention that the mechanism of action has a specific electrostatic origin. In addition, the appearance of a band at 1699 cm-1 in the benzamidine-inhibited form can be interpreted as resulting from the NC-N stretching vibrations of the amidinium moiety, which the observed crystal structure indicates is hydrogen-bonded to the carboxyl group of active-site Asp-189.


Subject(s)
Calcium/pharmacology , Trypsin Inhibitors/pharmacology , Trypsin/chemistry , Animals , Binding Sites , Cattle , Protein Structure, Secondary , Spectrophotometry, Infrared/methods , Trypsin/metabolism
3.
Am J Pathol ; 85(3): 555-68, 1976 Dec.
Article in English | MEDLINE | ID: mdl-998730

ABSTRACT

A significant number of offspring from brother-sister matings of NIH-Okamoto-Aoki spontaneously hypertensive rats (SHRs) were found to be normotensive at 20 weeks of age. Over 20% of the animals that were hypertensive at this age had mild-to-moderate unilateral hydronephrosis at the time of sacrifice. In over 90% of the rats that did not develop hypertension spontaneously, ligation of one ureter raised blood pressure above 150 mm Hg within 2 weeks. In those rats made hypertensive by obstructing one ureter and in those that developed hypertension with accompanying naturally occurring hydronephrosis, subcutaneous implants of fragmented renal medulla from unrelated normal rats decreased blood pressure to normotensive levels. In contrast, medullary implants had no significant effect in rats developing hypertension spontaneously without hydronephrosis. Renal inner medullary plasma flow was low in the obstructed kidneys of hydronephrotic hypertensive SHRs but was elevated in the kidneys of nonhydronephrotic hypertensive SHRs. The hypertension in hydronephrotic SHRs appears to be related to an impairment of the antihypertensive function of the renal medulla. Such an impairment of medullary antihypertensive function does not appear to play a significant role in the hypertension in SHRs without hydronephrosis.


Subject(s)
Hypertension/physiopathology , Kidney Medulla/physiopathology , Kidney/physiopathology , Animals , Hydronephrosis/complications , Hypertension/etiology , Hypertension/veterinary , Kidney/pathology , Kidney Cortex/transplantation , Kidney Medulla/blood supply , Kidney Medulla/transplantation , Ligation , Rats , Rodent Diseases/physiopathology , Transplantation, Homologous , Ureter/surgery
4.
Am J Physiol ; 231(5 Pt. 1): 1315-21, 1976 Nov.
Article in English | MEDLINE | ID: mdl-998774

ABSTRACT

To test the thesis that ureteral obstruction causes medullary ischemia, we determined inner medullary plasma flow (IMPF) in rats with bilateral or unilateral ureteral obstruction, and after relief of obstruction, by the intravenous 125I-albumin infusion technique. A progressive decline in IMPF was observed during obstruction of 18 h duration, greater in bilateral obstruction (7% of normal at 5h) than in unilateral obstruction (28% of normal at 5 h). The elevation in ureteral pressure was greater and more sustained in bilateral obstruction. After relief of obstruction, IMPF rose to 69-78% of normal in both groups within 2 h. Histologic studies showed tubular necrosis in portions of the inner and outer medulla immediately beneath the renal pelvic epithelium after bilateral or unilateral obstruction of 18 h duration, and India ink perfusion studies showed very poor filling of vasa recta in these areas. The concentrating defect in the postobstructive kidney may be related, at least in part, to damage inflicted by medullary ischemia during obstruction.


Subject(s)
Kidney Medulla/blood supply , Kidney/blood supply , Ureteral Obstruction/physiopathology , Albumins/metabolism , Animals , Blood Pressure , Body Water/metabolism , Female , Kidney Medulla/metabolism , Kidney Medulla/pathology , Kidney Papillary Necrosis/pathology , Osmolar Concentration , Pressure , Rats , Regional Blood Flow , Ureter/physiopathology , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathology , Ureteral Obstruction/urine
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