On the domain construction of the multienzyme gramicidin S synthetase 2. Isolation of domains activating valine and leucine.

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multienzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.

The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment.

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.

Induction of milk lipolysis by lipoprotein components of bovine blood serum.

The major blood lipid component responsible for activation of milk lipolysis was high density lipoprotein with density of 1.063 to 1.21 g . ml-1. Its low molecular weight apolipoprotein fraction, apo C, which activates milk lipoprotein lipase in vitro, was unable to induce milk lipolysis under normal conditions. Mechanical treatment of the milk rendered it highly susceptible to apo C-stimulated lipolysis. Low and very low density lipoprotein fractions, which also contain apo C, showed negligible effect on milk lipolysis. Apo C in combination with serum or heparin induced high lipolysis in normal milk. Also, lysolecithin influenced the degree of serum activated lipolysis. Antiserum raised against bovine apolipoprotein A-I, which does not activate lipoprotein lipase, removed the activating ability of serum. Induction of milk lipolysis is preceded by redistribution of lipoprotein lipase, thus increasing the accessibility of the enzyme to its substrates.

Isolation and characterization of lipoprotein lipase activators from bovine blood serum.

Bovine serum lipoproteins were fractionated into high density and low and very low density lipoproteins by precipitation with sodium phosphotungstate and magnesium chloride. From each lipoprotein fraction seven apo C peptides were isolated by gel filtration and ion exchange chromatography. The two lipoprotein fractions probably contain identical apo C peptides but in different proportions. Two of the apo C peptides activated lipoprotein lipase from milk in vitro. Their specific activities were about 2000 times as high as that of the original serum. The apo C fraction from low-very low density lipoproteins had a specific activity three times that from high density lipoproteins. Also, the activator peptides from low and very low density lipoproteins gave one band on alkaline urea gel electrophoresis whereas corresponding peptides from high density lipoproteins were slightly heterogeneous. The low and very low density lipoproteins, therefore, seem to be the fraction of choice for isolation of activators for lipoprotein lipase.

The fidelity of gramicidin S synthetase.

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.