John H. Copenhaver

CHARACTERIZATION OF ACIDIC PROTEINS IN CELL ACRYLAMIDE GEL ELECTROPHORESIS NUCLEI FROM RAT BRAIN BY HIGH‐RESOLUTION1

Abstract— Acidic proteins from cell nuclei of rat brain were evaluated by gel electrophoresis. By the use of a gel containing a high ratio of bis‐acrylamide to acrylamide, 31 bands were demonstrated for this protein fraction. Gels without the high concentration of copolymer lacked the resolving power to separate these proteins. Because the number of bands present in the acidic protein fraction is greater than the number of histones present in cell nuclei, the suggestion that the acidic proteins play a role in genetic regulation seems reasonable.

EXPERIMENTAL MATERNAL HYPERPHENYLALANINEMTA: DISAGGREGATION OF FETAL BRAIN RIBOSOMES

Disaggregation of polyribosomal structures has been demonstrated in fetal rat brains following treatment of the maternal animal with para‐chloro‐d,l‐phenylalanine (used primarily to inhibit maternal phenylalanine hydroxylase, EC 1.14.3.1) and with phenylalanine (used to raise the level of circulating phenylalanine in maternal and fetal plasma). Since highly disaggregated polyribosomal systems cannot support normal levels of protein synthesis in vitro, it has been postulated that the composition of the free amino acid pool(s) plays a regulatory role in protein synthesis through the intermediary effect of polyribosomal aggregation‐disaggregation. We believe that a possibly prolonged period of disaggregation of neuronal polyribosomes might disrupt neuronal protein synthesis sufficiently in utero to produce the mental insufficiency observed in the offspring of untreated maternal phenylketonurics.

Protein Metabolism in the Developing Brain: Influence of Birth and Gestational Age

Incorporation of carbon-14-labeled phenylalanine into brain protein of newborn pigs falls sharply within 24 hours after birth. This decrease is related to the time of birth rather than the gestational age of the piglets, although the latter is also associated with a gradual decrease in brain protein synthesis.

Daily variations in phenylalanine hydroxylase activity in male and female guinea pigs

Abstract Phenylalanine hydroxylase of guinea pig liver exhibited circadian rhythmicity in male animals, but not in female animals. Female guinea pigs showed variations in plasma phenylalanine and tyrosine which were similar to those observed over the same period for male animals. Reversal of the lighting cycle resulted in a marked change in cycles of plasma phenylalanine and tyrosine, but had little effect on the rhythm of hepatic phenylalanine hydroxylase activity. Peak phenylalanine hydroxylase activity in guinea pigs followed a few hours behind the peak of the rhythm of the phenylalanine/tyrosine ratio in plasma.

Quantitative analysis of plasma galactose and glucose by gas-liquid chromatography

Abstract A rapid, specific, and reproducible method is described for the determination of galactose and glucose in plasma or blood by means of gas-liquid chromatography. The use of trimethylsilyl ethers facilitates the separation and identification of the respective anomers of galactose and glucose on a Carbowax 20 M column. Several experiments are presented describing the effect of diet and injections of galactose on the blood levels of galactose and glucose in the adult female rat.

Inhibition by Phenylalanine of the Entry of 5-Hydroxy-Tryptophan-l-G14 into Cerebrospinal Fluid.

Summary High plasma levels of L-phenyl-alanine inhibit the entry of 5-hydroxytrypto-phan-C14 into the CSF of dogs. CSF is an accessible body fluid that may be used to study transport of amino acids into the CNS.

Effect of para-chlorophenylalanine on the growth and development of the fetal, neonatal, and adult rat

Abstract The chronic administration of para-chlorophenylalanine to maternal and young adult rats resulted in an immediate reduction in food consumption and a concomitant loss in body weight. Similar effects were observed in fetal and neonatal animals. It is suggested that a causal relationship exists between the hunger response(s) and para-chlorophenylalanine. Chlorophenylalanine was evaluated with respect to various parameters related to growth and development of the rat at different ages.

Quantitation of stained proteins in polyacrylamide gels

At present, techniques which are used for the quantitative analysis of protein bands in polyacrylamide gels following electrophoresis are: scanning the unstained gel in an ultraviolet monitor at 280 nm (1) ; scanning a stained gel in the visible region (1) ; slicing out the stained protein bands followed by a spectrometric analysis of the solubilized dye (2) ; photographing the stained protein bands and using a densitometer to scan the negative (3). Rlost of these techniques have certain inherent disadvantages depending on instrument sensitivity, time involved for a complete assay, gel pattern and length of gel column. For example, in the first technique, the main disadvantage of scanning unstained gels at 280 nm is the baseline noise. In the third method if the resolution of the protein bands is incomplete or if the protein bands are skewed the ability to slice out individual bands is impossible. In addit’ion, removal of all the dye from the acrylamide slices may take much longer than 2 hr at 50” (unpublished results). The accuracy of the fourth method is contingent on how well the gel can be photographed and type of film used. In an effort to eliminate some of the disadvantages inherent in the above techniques, we hare developed a method for the quantitation of protein bands in polyacrylamide gels. This method involves scanning protein gels at 280 nm which have been previously stained with naphthol hlue black. The sensitivity of detection of stained protein bands \vas found to surpass unstained protein bands when scanned at 280 nm and, the problem of protein diffusion has been eliminated since the proteins are fixed in 7%) acetic acid. Also reported is a scanning device \vhich can he constructed from instruments found in most laboratories. The ap-

ENTRY OF L‐[14C]PHENYLALANINE AND α‐AMINO[14C]ISOBUTYRIC ACID INTO THE CEREBRO‐ SPINAL FLUID AND BRAIN OF DOGS*

THE STUDY of entry of isotopically-labelled amino acids into the central nervous system and their incorporation into protein has been the subject of much interest in recent years (WAELSCH and LAJTHA, 1961 ; LAJTHA, 1964). One major difficulty in the interpretation of data from analysis of brain homogenates is the special complexity of brain substance in terms of differences of cell type and of regional variation. This laboratory has been interested in cerebrospinal fluid as a homogeneous central nervous system fluid whose accessibility allows in vivo study of blood-brain barrier transport systems. Previous studies established that factors affecting transport of a labelled amino acid into the brain can be detected by examination of the CSF during the living state (SCHAIN, COPENHAVER and CARVER, 1965). The present investigation is concerned with the study of the entry of isotopicallylabelled amino acids into CSF as compared to entry into several anatomically distinct brain regions. Preliminary investigations indicated that there were similarities in the time sequence of entry of labelled amino acids into CSF and brain substance (CARVER, SCHAIN and COPENHAVER, 1966). However, interpretation of studies with single injections presents difficulties because of the uncertainties imposed by variable and rapidly changing blood levels. In these experiments, blood levels of the labelled amino acids were maintained by use of a constant infusion pump. An inert unnatural amino acid (cx-aminoisobutyric acid) and a relatively slowly metabolized natural amino acid (phenylalanine) were chosen for adminstration. Unanticipated differences were found in the patterns of entry of these two substances into the CNS.

A fluorometric procedure for estimation of galactose-1-phosphate uridylyltransferase activity in red blood cells.

A simple, rapid, quantitative procedure has been developed for the estimation of galactose-1-phosphate uridylyltransferase activity in RBC hemolyzates. The method is based on the native fluorescent emmission spectra of NADPH formed by d-glucose-6-phosphate: NADP oxidoreductase and 6-phospho-d-gluconate: NADP oxidoreductase present in the hemolyzate. This procedure offers the advantages of small sample size with low blank readings. No subsequent product isolation or conversion to fluorophores is necessary. The fluorometric procedure is applicable over a wide range of NADPH concentrations and the fluorophore is stable for a period of hours. The procedure has been employed to measure the transferase activity in RBC hemolyzates obtained from both humans and rats.