Faculty Bibliography

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Masimal activity against hemoglobin was obtained at pH 3.2-3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus

The degradation of delta sleep inducing peptide (DSIP) was studied and compared with that of Met-enkephalin in brain homogenates. After 7.5 minutes of incubation of 2.5% (w/v) homogenate, 30% of N-terminal tryptophan was released from DSIP, while 75% of N-terminal tyrosine was released from Metenkephalin. The optimal pH of degradation of both peptides was pH 7.35. A rapid increase of breakdown of the peptides was observed during the 10 days of postnatal growth, with breakdown expressed per unit weight of brain. Neither morphine nor deprivation of rapid eye movement (REM) sleep affected the degradation of peptides

In a study of a system suitable for investigating long-term effects on brain protein metabolism, we measured amino-acid incorpration into isolated immature brain explants incubated under sterile conditions up to ten days. Measurements of changes in total proteins, total DNA, cell number during the experiments, and 14C-thymidine incorporation measurements indicated no significant net growth; new cell formation was below 5% in a 5-day period; therefore, amino-acid incorporation was mainly due to protein turnover. The rate of incorporation in our immature brain preparation was similar to that of the adult brain in vivo: by ten days about one-half of the tissue protein turned over. The label incorporated was released in subsequent incubations with cold amino acids. Such release occurred in all subcellular fractions examined. Incorporation was fairly stable; at temperatures below 30 degrees C it rapidly declined, but it was not affected when phenylalanine or the branched chain amino acids (leucine, isoleucine, valine) were elevated in the incubation medium. Brief exposure to low amino-acid media had no effect; longer exposure resulted in tissue damage. Our model system indicates that overall brain protein turnover is not sensitive to such variations in the level of most amino acids, which may occur under various conditions. Protein metabolism of the nervous system occurs at a high rate. A recent long-term labeling method (Lajtha, Latzkovits, and Toth, 1976) gave a best fit to incorporation curves by assuming two compartments for adult brain proteins, one of which (about 6%) has a half-life of 15 hr and the other (94%) has a half-life of ten days. The disappearance of protein-bound label with time under conditions in which all proteins were previously labeled indicated that most, possibly all, proteins in brain are in a dynamic state (Lajtha and Toth, 1966). Incorporation of amino acids was found in all proteins and structures that have been studied to date; myelin proteins previously thought less active are also metabolized at a significant rate (Sabri, Bone, and Davison, 1974; Lajtha, Toth, Fujimoto, and Agrawal, 1977). We have fairly extensive information available in addition to turnover studies about the mechanisms of protein synthesis in brain (Roberts, 1971); protein breakdown was also studied in some detail (Marks and Lajtha, 1971). In contrast to our knowledge about protein metabolism under physiological equilibrium conditions, our information about alterations during functional demands or pathological conditions is scanty. Although a significant amount of work has been reported, largely because of technical difficulties the results are difficult to interpret unequivocally. The present report represents our effort to address some of the obstacles: to develop a system in which influences on long-term incorporation can be studied..

When rats are put on a diet that is low in protein or contains no protein, decrease in brain weight can be observed. Changes in adults are minimal. In the young there is a 10--30% decrease in cell number and protein content; the cell size (protein per cell) does not change significantly. The change is greater, the earlier the diet is started and the more severe the protein dificiency is. The longer the malnutrition period lasts, the smaller is the recovery to normal values on subsequent control diets. Amino acid incorporation in the brain decreased 10--30% under these experimental conditions; it seems the decrease was to a great extent in the more slowly metabolized protein pool. Changes in other organs were greater; for example, in liver the decrease was up to 75% under similar conditions. The changes in the brain were heterogeneous; there were regional differences, and not all proteins were affected to the same degree; choline acetyltransferase was not affected. Cellular amino acid transport as studied with incubated slices of brain was not altered under these conditions

Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (PheH) to activated CH-Sepharose-4B. When 12 mg of ligand was reacted with 100 mg of matrix, 20% of the initial enzyme activity was covalently bound along with 55% of the protein. The coupled enzyme showed greater thermal stability from 50 degrees to 60 degrees after heating for 15 min, a lower optimum pH, 5.8, slightly less inhibition by Ag+, Cu+2, and Hg+2, and greater resistance to hydrolysis by alpha-chymotrypsin and protease. The uncoupled enzyme, however, exhibited greater storage stability than the covalently linked enzyme at 25 degrees after 24 hrs and at 0 degrees after 21 days. Alteration of the microenvironment by the introduction of sulfhydryl groups and positive and negative charged carriers during coupling of the enzyme either had no effect or markedly reduced hydroxylase activity

A decrease in amino acid influx and exit in incubated slices when the temperature was lowered from 37 to 20°C was observed with all 16 amino acids examined at two concentrations (1 mM and 10 μM). The temperature dependence of cellular amino acid influx observed in slices in vitro contrasts with the absence of temperature dependence of capillary amino acid influx in the brain in vivo that we recently reported. The temperature effects in slices varied some-what among the various amino acid transport classes. With some amino acids (Phe, Val, Tyr, Asp, Glu), especially at lower concentrations, the greater inhibition of exit than of uptake resulted in an actual increase in tissue amino acid uptake at lower temperatures in long-term experiments. The results indicate heterogeneity in the effect of temperature on the various transport classes and show a difference between capillary and cellular amino acid transport in the brain.