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Background: The DoloTest is a newer health-related quality of life (HRQoL) monitoring instrument for pain, not yet validated for headache. Aims: To examine the usefulness of the DoloTest in a specialized headache center. Methods: The sample consisted of patients referred to psychologists from the Danish Headache Center (DHC) for whom the test was carried out at start of, end of, and 6 months after treatment. Points on eight scales of the test were measured (values ranged from 0 to 100), then totaled (0 to 800). Scores were analyzed using Wilcoxon Signed Ranks test. The correlation between headache days and DoloTest scores were computed using linear regression adjusted for age. Qualitative feedback on usefulness of the test were gathered from psychologists. Results: Of 135 patients included, 105 completed treatment. On average, headache days decreased from 22 days per month at start of treatment (SD 9.0, median 29) to 18 days at end of treatment (SD 10.8, median 19) (p < 0.001). At end of treatment, DoloTest scores improved for pain (p = 0.015) and reduced energy and strength (p = 0.034). At 6 months' follow-up, total scores improved (p = 0.034), as well as component scores for pain (p = 0.010), problems with strenuous activity (p = 0.045) and reduced energy and strength (p = 0.012). Correlation between reduced headache days and improved DoloTest scores was 0.303 (p = 0.028). Psychologists found the test useful in monitoring and evaluating patients. Conclusions: The DoloTest was useful for psychoeducation and for monitoring the effect of headache treatment. Implications: The DoloTest is a potential HRQoL monitoring instrument for headache patients. We recommend further validation studies. [ABSTRACT FROM AUTHOR]

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments. [ABSTRACT FROM AUTHOR]

The amino acid analog L-cyclohexylalanine, which may be considered an analog of the hydrophobic amino acids leucine and valine, was found to thioester-bind to the heavy enzyme of gramicidin S synthetase. The results indicate that it preferably binds to the thiol site of leucine, although binding to the valine site also occurs. In the synthesis of the cyclic decapeptide by gramicidin S synthetase, the results suggest that L-cyclohexylalanine can replace L-leucine and L-valine. We have also found that in the synthesis of the cyclic decapeptidase L-leucine can replace L-valine and vice versa. The results further indicate that the previously reported thioester binding of D and L-phenylalanine to the heavy enzyme takes place at the thiol site of leucine. [ABSTRACT FROM AUTHOR]

When a mixture of the light enzyme of gramicidin S synthetase containing thioester-bound D-[14C]phenylalanine and the heavy enzyme is passed through a Sepharose column containing carboxyl-bound proline (substrate amino acid) as ligand, the two enzymes are retained as the result of the affinity between the heavy enzyme and the ligand and the affinity between the two enzymes. Upon elution and separation of the two enzymes it was demonstrated that the D-[14C]phenylalanyl groups had been transferred to the heavy enzyme. This transfer did not take place when similar experiments were carried out using as ligands valine, ornithine or leucine which are also substrates for the heavy enzyme. Hence binding of proline to the heavy enzyme is required for transfer to take place. Upon incubation of the heavy enzyme containing the transferred D-[14C]phenylalanyl group with the light enzyme, ATP and the five unlabelled substrate amino acids, radioactivity is incorporated into gramicidin S. This suggests that in the biosynthesis of gramicidin S, the D-phenylalanyl group is transferred to an acceptor site on the heavy enzyme and subsequently to the imino group of thioesterbound proline. In view of what is known about gramicidin S synthesis it seems likely that the acceptor site is a thiol site. [ABSTRACT FROM AUTHOR]

Columns containing one of the amino acids in gramicidin S bound through its carboxyl group to the amino group of amino-alkyl-Sepharose (CNBr-activated Sepharose + 3,3′-diaminodipropylamine) have been prepared and were found to exhibit affinity for the corresponding enzymes (light or heavy) of gramicidin S synthetase. The D-phenylalanyl ligand will bind the light enzyme and the L-propyl, L-valyl, L-ornithyl or L-leucyl ligand, the heavy enzyme. These columns permitted a study of the interaction of the light and heavy enzyme. The results show that the area of the light enzyme which has affinity for the heavy enzyme is that part which activates phenylalanine. The area of the heavy enzyme which interacts with the light enzyme is not that part of the enzyme which activates proline. However, it is probably located close to the activation sites for proline and leucine. The columns, in particular with proline as ligand, provide a quick and convenient method for the separation of the light and heavy enzyme of gramicidin S synthetase. [ABSTRACT FROM AUTHOR]

The results show that enzyme I of gramicidin S synthetase activates D-leucine in a manner analogous to that of L-leucine (ATP + D-leucine ↔ D-leucyl-adenylate +. PPi) and binds two moles of D-leucine in thioester linkage. It has therefore two thiol sites for D-leucine, compared to one for the L-isomer. One of the sites seems to be the thiol site for L-leucine which is therefore stereo-unspecific. The results also indicate that the site for the formation of the aminoacyl adenylates is the same for the two isomers. In contrast to thioester-bound L-leucine, enzyme-bound D-leucine does not catalyze the ATP-[14C]AMP exchange reaction. At a molar ratio of L-leucine: D-leucine equal to 4, gramicidin S synthesis is almost completely d and the growth of the peptide chain stops at the tetrapeptide stage (D-Phe-L-ProL-Val-L-Orn). Enzyme-bound D-leucine is thus not able to replace L-leucine in the formation of the pentapeptide. [ABSTRACT FROM AUTHOR]

Kinetic studies show that AMP inhibits the activation of ali the amino acids participating in the formation of gramicidin S. The activation was measured by the amino-acid-dependent ATP-[32P]pyrophosphate exchange reaction. AMP acts as a competitive inhibitor to ATP and a non-competitive inhibitor to the amino acids. The large difference in the effect of AMP on the activation of L-phenylalanine and D-phenylalanine makes it probable that the activation of the two isomers takes place on different sites on the enzyme. [ABSTRACT FROM AUTHOR]