STUDiES ON THE TURNGVER 0F PROTEEN, GLYCOPROTEEN AND GANGLEOSEDES EN THE BRAENS 0F . GALACTOSE ENTDXECATED CHECKS Thesis for the Degree of .Ph. D. MICHIGAN STATE UNWERSITY JAMES CARLISLE BLOSSER 1 9 72 'YHPR:I This is to certify that the thesis entitled STUDIES ON THE TURNOVER OF PROTEIN, GLYCOPROTEIN AND GANGLIOSIDES IN THE BRAINS OF GALACTOSE INTOXICATED CHICKS presented by JAMES CARLISLE BLOSSER has been accepted towards fulfillment of the requirements for M—Jegree in My Date 2/2h/72 0-7639 LIBRARY Michigan Slim University c amomc av .1 Bouxamnmmc. { LIBRARY BINDERS ‘ sIRIIGPOEIT. menial; ABSTRACT STUDIES ON THE TURNOVER OF PROTEIN, GLYCOPROTEIN AND GANGLIOSIDES IN THE BRAINS OF GALACTOSE INTOXICATED CHICKS BY James Carlisle Blosser A study of the effects of feeding chicks tonic levels of D-galactose on brain amino acid levels, poly- ribosomal aggregation, protein turnover, glyc0protein, ganglioside and mucopolysaccharide synthesis and neural lysosomal stability was undertaken. A number of amino acids known to be associated with the tricarboxylic acid cycle were significantly altered in concentration; alanine, glutamate, and glutamine were decreased while asparate levels increased. In addition, leucine concen- trations were depressed. Brain polyribosomal profiles were similar to those from controls. The ratio of mono- ribosomes to polyribosomes was 0.10 i 0.01 while yields of ribosomal material were typically 0.50 mg per gram of brain. Protein synthesis rates, as judged by the in viva 1 incorporation of L-[U- 4C] leucine and L-[guanidino-14C] arginine, were not decreased in whole brain or in the James Carlisle Blosser nuclear, microsomal, mitochondrial, or soluble fractions. Protein degradation rates, as measured by the in_gi!g loss of L-[guanidino-14C] arginine, were similar to those measured in controls. Microsomal protein had the longest half—life (60 hours) of the subcellular fractions studied. The average half-life for total protein was 36 hours. Both [6-3H] glucosamine and [6-3H] mannosamine were incorporated into glycoprotein and ganglioside fractions at enhanced rates in galactose-fed chicks. The increased utilization of [3H] glucosamine for glycoprotein synthesis was greatest in the microsomal fraction and typically 50 per cent greater than controls over the first 30 minutes. Free glucosamine concentrations from galactose treated animals were similar to those in con- trols (2.2 i 0.1 and 1.9 i 0.1 nmoles/gram tissue, respectively) as were levels of glycoprotein bound hexosamine. Approximately 80 per cent of the tritium was incorporated as hexosamine into the glycoprotein fraction. In similar analyses of the ganglioside fraction, neutral and amino sugar contents were unaltered from those of control chicks and radioactivity was found to be distributed between hexosamine (70%) and sialic acid (30%). [BB] Mannosamine was incorporated into glyc0pro- tein of galactose-fed chicks at three times the rate of that in control animals and radioactivity was solely in sialic a side fral faster t than in into chc of acid cursors an enha: remain % demonstfl tempera: increaSE Preincu: tOSe an: iRCUCed, by remoy accompa: t°5€ an; (00n-s€d and B‘Na controls to the S centratil l'l‘lcreasE I aCCumula James Carlisle Blosser sialic acid. Similar results were obtained in the ganglio- side fraction. Although [3H] glucosamine was taken up faster by mucopolysaccharides in galactose-fed chicks than in controls, normal incorporation rates of 35504 into chondroitin sulfate suggests that this subclass of acid polysaccharides are not affected. That pre- cursors to the carbohydrate moieties are utilized at an enhanced rate while the content their bound products remain unaltered is suggestive of a faster turnover rate. Neural lysosomes from galactose-fed chicks demonstrated decreased stability to hypoosmotic and temperature shock when compared with controls. This increased lability to osmotic shock could be duplicated by preincubation of normal lysosomes in solutions of galac- tose and galactitol. Further, the increased fragility induced, in yiyg, by galactose feeding could be reversed by removing the diet from the chicks for 8 hours, and was accompanied by large reductions in the levels of galac- tose and galactitol in the brain. The free activities (non-sedimentable at 20,000 g) of both B-galactosidase and B-Nacetylhexosaminidase were elevated above those of controls, and the increase was found to be proportional to the summation of brain galactose and galactitol con- centrations. Together, these data indicate that increased fragility of lysosomes is a result of the accumulation of galactose and galactitol by the lysosori a resul hydrola. l | concept of glyc. mechanic patient; brain d; James Carlisle Blosser lysosomes. The possibility of lysosomal dysfunction as a result of increased lability and release of acid hydrolases into the cytoplasm is consistent with the concept of a faster turnover rate of carbohydrate units of glycoprotein and gangliosides. Whether a similar mechanism operates in the brains of galactosemia patients contributing to their frequent irreversible brain damage is now open to further exploration. STUDIES ON THE TURNOVER OF PROTEIN, GLYCOPROTEIN AND GANGLIOSIDES IN THE BRAINS OF GALACTOSE INTOXICATED CHICKS BY James Carlisle Blosser A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 The Wells for 1 support, a: Although n for their 0f critica Single out P. Kozak f and Constx free atmos aSSOCiates 'i‘Ppreciat£ gratitude enCOuI-age] / vl‘ ) (1b (97 ACKNOWLEDGMENTS The author wishes to thank Professor William W. Wells for his challenging ideas, constant optimistic support, and more than generous financial assistance. Although numerous other individuals could be mentioned for their roles in facilitating my growth in develOpment of critical thought and reliable values, I would like to single out in particular Drs. Harvey H. Knull and Leslie P. Kozak for their unselfish attention, honest criticisms, and constructive discussions. The friendly and tension- free atmosphere of the laboratory created by the research associates and fellow graduate students has also been appreciated. Finally, I would like to express my deep gratitude to my parents, whose unselfish love and encouragement has allowed this venture to be undertaken. ii List of List of List of Chapter I. TABLE OF CONTENTS Tables 0 O O O O O O O O O O O 0 Figures I O O O O O O O O O O 0 Abbreviations. . . . . . . . . . . INTRODUCTION . . . . . . . . . . . Organization of the Thesis. . . . . . Research Objectives and Rationale of Experimental Approach . . . Literature Review . . . . . Human Hereditary Galactosemia . . . symptoms 0 O O I I O O O O Genetics . . . . . Galactose Metabolism in Galacto- semics O O O O O O O O I Comparison of the Rat and Chick as Model Systems . . Possible Roles of Galactose, Galactitol and Galactose-l- Phosphate in the Etiology of Galactose Toxicity Syndrome . . . . . . . . . . Cataract Formation in the Lens . . Aminoaciduria . . . . . . . Biochemical Changes in the Brain. . Galactose Toxicity in Cell Cultures and Red Blood Cells . . . . . References . . . . . . . . . . . iii Page vi vii ix 12 12 l3 l3 16 18 Chapter II. III. STUDIES ON AMINO ACID LEVELS AND PROTEIN METABOLISM IN THE BRAINS OF GALACTOSE INTOXICATED CHICKS . O O O O O O 0 Abstract 0 I I O O O O O O O 0 Introduction . . . . . . . . . Materials and Methods . . . . . . . Animals and Materials . . . . . . In_vivo Tracer Studies. . . . . . Preparation of Samples for Counting . Determination of the Specific Radio- activity of Free [14C] Leucine . . Preparation of Tissue for Free Amino Acid Quantification . . . . . . Polyribosomal Profiles. . . . . . Determination of Protein Bound Hexosamine . . . . . . . . . Estimation of the Specific Radio- activity of Free [3 H] Glucosamine . Results 0 O O O O O O O O O O O [14 Polyribosomal Profiles and Rates of Synthesis of Nascent Polypeptide C] Leucine Incorporation In Vivo Into Brain Protein . . . . . Chains . . . . . . . . . In Vivo Catabolism of Brain Protein . B1os ynthesis of Glycoprotein. . . . Discussion . . . . . . . . . . Acknowledgments . . . . . . . . . References . . . . . . . . . . BIOSYNTHESIS 0F GLYCOPROTEIN, GANGLIOSIDES AND MUCOPOLYSACCHARIDE IN BRAINS OF CHICKS FED D‘GALACTOSE o o o o o o 0 Introduction . . . . . . . . . Materials and Methods . . . . . . . Animals and Materials . . . . . . I2_Vivo Tracer Studies. . . . . . Isolation of Subcellular Fractions. . Isolation and Determination of the Specific Radioactivity of Glyco- proteins. . . . . . . . Isolation and Determination of Radio- activity in Mucopolysaccharides . . iv Page 24 24 25 26 26 27 28 29 3O 30 31 31 34 34 35 36 37 38 43 45 57 57 58 58 59 6O 6O 61 Chapter Isolation of Gangliosides . . . . . Quantitation and Specific Radio- activity Determination of Sialic Acid in Gangliosides . . . . . . Quantitation of Bound Hexosamine and Hexose. . . . . . . . Isolation of Free [3H] Mannosamine . . Results . . . . . . . . . . . . Incorporation of [3H] Glucosamine Into Gangliosides . . . . . . . Biosynthesis of Mucopolysaccharides . Incorporation of [3H] Mannosamine Into Glycoprotein and Gangliosides . Discussion . . . . . . . . . . . References . . . . . . . . . . . IV. ENHANCED FRAGILITY OF NEURAL LYSOSOMES FROM CHICKS SUFFERING FROM GALACTOSE TOXICITY O O C O O O O O O O O 0 Abstract . . . . . . . . . . Introduction . . . . . . . . . . Materials and Methods . . . . . . . Animals and Materials . . . . . . Preparation of Lysosomal Fraction . . Enzymatic Assays . . . . . . . Quantification of Galactose and Galactitol . . . . . . . . . ReSUlts O O O O O O O O O O O 0 Stability of Lysosomes to Osmotic and Temperature Shock . . . . . . Stability of Lysosomes Preincubated in Galactose or Galactitol to Osmotic Shock . . . . . . . . . Activities of Free Acid Hydrolases as a Function of In Vivo Levels of Galactose and Galactitol. . . . . Discussion . . . . . . . . . . . References . . . . . . . . . . . V O SUMMRY O O O O O O O O O O O I O Page 62 62 63 64 65 65 65 66 67 73 89 89 9O 91 91 92 92 94 94 94 95 97 99 105 118 LIST OF TABLES Table Page 1. Analysis of Free Amino Acids and Ammonia in the Brains of Chicks Fed Control or Galactose-Containing Diets . . . . . . 47 2. Incorporation of L-[U-14C] Leucine into Brain Subcellular Fractions . . . . . . . 48 3. Levels of Neutral and Amino Sugars in Ganglio- sides, Glycoprotein and Mucopolysacchae' rides O O O O O O O O O O O O O 75 4. Change in Radioactivity in Free Mannosamine and the TCA Soluble Fraction with Time Following Intracerebral Injection of 1 uCi of [6-3H] Mannosamine . . . . . 76 5. Ig_vivo Levels of Galactose and Galactitol and Free Activities of B-Galactosidase and B-N-Acetylhexosaminidase in Brains of Chicks Fed a High Galactose Diet . . . . 107 6. Soluble Activities of B-Galactosidase and B-N-Acetylhexosaminidase in Brains of Chicks Fed Control or Galactose-Containing Diets . . . . . . . . . . . . . 108 7. Levels of Selected Acid Hydrolases in Plasma . 109 vi Figure 1. 2. 10. 11. 12. 13. LIST OF FIGURES Major Metabolic Pathways of D-Galactose . . . Incorporation of L-[U-14C] Leucine into Pep- tidyl tRNA on the Polyribosomes . . . . Polyribosomal Profiles from two Typical Exper— iments are Shown. . . . . . . . . . In vivo Rates of Protein Degradation as Illus- trated with L-[guanidino-14C] Arginine . . Incorporation of D-[6-3H] Glucosamine and L- [guanidino-14C] Arginine into the Different Subcellular Fractions of Galactose- and Control-fed Chicks . . . . . . . . . Incorporation of [3H] Galactosamine into Gangliosides . . . . . . . . . . . Incorporation of [3H] Hexosamine into Mucopoly- saccharides . . . . . . . . . . . 35804 Incorporation into Chondroitin Sulfate . [3H] Mannosamine Incorporation into Glycopro- tein as Sialic Acid . . . . . . . . [3H] Mannosamine Incorporation into Ganglio- sides as Sialic Acid . . . . . . . . Incorporation of [3H] Mannosamine into Glyco- protein of Subcellular Fractions . . . . Effect of Osmotic Shock on Stability of Lyso- somes from Chick Brains . . . . . . . Effect of Increasing Temperature on Stability of Neural Lysosomes. . . . . . . . . vii Page 23 50 52 54 56 78 80 82 84 86 88 111 113 Figure 14. Osmotic Stability of Neural Lysosomes Pre- incubated in Galactose, Galactitol, or Sucrose . . . . . . . . . . . 15. Reversal of Neural Lysosomal Fragility Induced by the Galactose Diet. . . . viii Page . 115 . 117 UDPAG CMP TCA UDP Tris ATP EDTA Pi PPi DPM CPM ABBREVIATIONS uridine diphospho-N-acetyl glucosamine cytidine monophosphate trichloroacetic acid uridine diphOSphate tris (hydroxymethyl) aminomethane Adenosine triphosPhate Ethylenedinitrilotetraacetic acid Adenosine diphosphate inorganic phosphate inorganic pyrophosphate decompositions per minute counts per minute ix CHAPTER I INTRODUCTION Organization of the Thesis For convenience, Chapters II, III, and IV have been organized in article form as found in biochemical journals. Each chapter includes an introduction, methods, results, and discussion section. A bibliography is pro- vided at the end of each chapter. Chapter II has been published as it appears under the title, "Studies on Amino Acid Levels and Protein Metabolism in the Brains of Galactose-Intoxicated Chicks" by J. C. Blosser and W. W. Wells, Journal of Neurochemistry, 19, 69 (1972). Chapter IV, with omission of the data on plasma levels of acid hydrolases, has been accepted for publication in the Journal of Neurochemistry under the chapter title by J. C. Blosser and W. W. Wells. Abstracts for these two manuscripts are included in the text of the thesis. Research Objectives and Rationale Of Experimental Approach Although the neuropathological consequences of galactosemia can be largely avoided therapeutically by restriction of dietary galactose, the underlying bio- chemical causes of mental retardation are unknown. Research in this area, by implementation of model systems, is designed to elucidate the altered bio- chemical parameters contributing to motor and sensory dysfunction and will hOpefully lead to an understanding of the biochemical causes of impaired mental function in the human galactosemic. Such altered factors may also be common to a number of other known inherited mental disorders, for example the amino acidopathies, and hence contribution could be made to an understanding of their etiologies. Chicks appear to be a useful model for studying the galactose toxicity syndrome due to the low levels of the sugar nucleotide pathway enzymes in brain tissue and their high susceptibility to the neu- rotoxic effects of galactose. The initial observation of depressed levels of ATP and phosphocreatine and energy charge in the brains of galactose-fed chicks (Kozak and Wells, 1969) prompted a consideration of the consequential effects on anabolic metabolism. Altered amino acid levels (Kinoshita, gt_§l., 1965) and decreased protein synthesis (Dische, Zelmenis, and Youlous, 1957) were known to occur in the lenses of galactose-fed rats, giving further impetus to under- take a study of protein turnover and its possible rate limiting mechanisms in the brains of galactose-fed chicks. In yiyg_radioactive tracer studies with leucine and arginine indicated that protein turnover in general was not affected and an investigation of protein subclasses was then begun by studying glycoprotein synthesis. [3H] glucosamine was found to be incorporated at an enhanced rate into the glycoprotein, glycolipid, and mucopoly- saccharide fractions. Because of the ambiguity in interpretation of tracer studies of this sort, other labeled precursors of these glycomacromolecules were used to verify the above observations. If any or all of these fractions are being synthesized at a greater rate than normal, any precursor should be utilized at a faster rate. Sodium [358] sulfate incorporation rates into the mucopolysaccharide fraction was normal, but [3H] mannosamine was incorporated into glycoprotein and glycolipid as N-acetylneuraminic acid much faster than in control animals. Since levels of glucosamine, galac- tosamine, and N-acetyl neuramimic acid in the glycolipid and glycoprotein of whole brain were essentially unaltered, the possibility of a faster turnover rate was considered. Levels of B-N-acetylhexosaminidase and B-galactosidase, both lysosomal enzymes, were found to be elevated in post lysosomal supernatant fractions of tissue homo- genates and subsequent studies demonstrated enhanced fragility of the neural lysosomes as a result of accumulation of galactose and galactitol in the brain. These findings are consistent with the hypothesis of a faster turnover rate of glycoprotein and/or glycolipid. Literature Review Galactosemia is an inherited autosomal recessive disease characterized by the inability to metabolize D—galactose. A myriad of clinical manifestations of the disorder including liver disease, cataracts, and mental retardation are the result of a deficiency of galactose-l- phosphate uridyltransferase [E.C. 2.7 7.12] activity (Isselbacher, Anderson, Kurahashi and Kalckar, 1956). The purpose of this review is to briefly describe the clinical, genetic, and biochemical characteristics of the disease, compare the animal model systems used for studying the galactose toxicity syndrome, and describe the roles suggested for galactose and its metabolites in contributing to the pathological effects of galactose feeding in animals and cell cultures. Human Hereditary Galactosemia Symptoms.--The symptoms associated with galacto- semia usually appear shortly after birth and are casually related to the ingestion of galactose in the form of lactose in the mother's milk. The most common initial symptom is a failure of the infant to thrive and is characterized by a loss of weight, refusal to eat, lethargy, and the development of diarrhea and vomiting. Jaundice is cirrhosis c are observa of milk cor. proteinurie and occasic wide range anumber 01 at times b} shortly the hiindividi life. but i IEtardatio; Al Cm‘be rev the infant degree of to range v firSt Ye?” USUally he problems Jaundice is a common occurrence and often proceeds to cirrhosis of the liver. Cataracts and mental retardation are observable within the first two months if ingestion of milk continues. Other symptoms include amino aciduria, proteinuria, and galactosuria reflecting renal malfunction and occasionally hypoglycemia. There appears to be a wide range of variation in severity of the symptoms. In a number of cases the disease is fulminating, accompanied at times by convulsive activity, with death ensuing shortly thereafter. Galactosemia has also been diagnosed in individuals with relatively mild symptoms in early life, but who often are found to suffer from mental retardation in later life (Isselbacher, 1966). All the above symptoms except mental retardation can be reversed if the disease is diagnosed quickly and the infant is placed on a galactose free diet. The degree of mental retardation, as judged by IQ, appears to range widely when dietary therapy is started by the first year of life. Those with normal IQ are found but usually have special learning defects and psychological problems (Sadler, Inouye and Hsia, 1969; Komrower and Lee, 1970). The highest IQ's reported as a group are those of children whose mothers restricted lactose ingestion during the child's prenatal life (Donnell, Koch and Bergren, 1969). At present, no therapy or cure for galactosemia is available beyond dietary control. The recent demon- stration of lambda phage transduction of a galactose operon into the DNA of cultured human galactosemic fibroblasts and the resultant expression of galactose-1- phOSphate uridyltransferase activity in the cell, suggests the possibility of introducing the gene to cells in vizg_which lack transferase activity (Merril, Geier and Petricciani, 1971). Successful introduction of the gene into, say, liver cells might allow galacto- semics to thrive unaffected on normal or partially restricted diets. Genetics.--Numerous investigations of galactose-l- phosphate uridyl transferase activity in red and white blood cells in family lives have shown that galactosemia is transmitted by an autosomal recessive gene. Hetero- zygotes normally have 50 per cent of normal activity (reviewed by Segal, 1971). In at least one case the genetic defect appears to be due to a point mutation. Antibody to the normal transferase enzyme was shown to be quantitatively absorbed to galactosemic erythrocyte preparations which contained no transferase activity (Tedesco and Mellman, 1971). This is consistent with the findings by Beutler (1965) of several individuals whose red blood cell enzyme levels were 50 per cent of normal but whose parents had levels 75 per cent of the normal activity. Depending upon the location of a point mutation, the resultant structural change in the enzyme could moderately reduce enzymatic turnover rate or render the enzyme completely inactive. Galactose Metabolism in Galactosemics.-—The uridine nucleotide pathway for conversion of galactose to glucose has been largely eludicated by Leloir (1951) and is repre— sented by Steps 1 through 3 in Figure 1. Absence of galactose-l-phosphate uridyl transferase (reaction 3) blocks the major metabolic route for galactose and other oxidative and reductive pathways become more prominent. Galactose-l-phosphate is the initial metabolite to accumulate before the metabolic block. Elevated levels have been measured in red blood cells, liver, and kidney of galactosemics (Sidbury, 1960). However, both substrate and product inhibition of galactokinase (Figure 1, reaction 1) (Cuatrecases and Segal, 1965) as well as phosphatase activity (Figure 1, reaction 4) would tend to decrease the formation of this intermediate, a possible toxic metabolite. The reduction of galactose by aldose reductase (Figure 1, reaction 7) to galactitol was demonstrated when the polyol was detected in urine and plasma of a galactosemic patient (Wells, Pittman and Egan, 1964). Subsequent studies (Quan-Ma, H. Wells, W. Wells, Sherman and Egan, 1966) with tissues of two infants with galactosemia who died from the illness showed accumulation of galactitol in all tissues with highest concentrations in brain and skeletal muscle where the enzyme has highest l4C] galactose in a activity. Tracer studies with D-[l- galactosemic patient further demonstrated the major significance of this pathway in the absence of trans- ferase activity (Egan and Wells, 1966). However, due to the relative high Km (15 mM) (Hayman and Kinoshita, 1965), this pathway is active only when high levels of galactose are present. Galactose oxidation to galactonic acid by galac- tose dehydrogenase (Figure 1, reaction 8) followed by decarboxylation to xylulose (Figure 1, reaction 9) has been described in rat liver (Cuatrecasas and Segal, 1966). Although tracer studies with [1- or 2-14C] galactose suggested an insignificant role for this pathway in normal man, similar studies in galactosemics indicate that this metabolic route may play a role in galactose metabolism (Segal and Cuatrecasas, 1968). Another pathway for galactose metabolism which could bypass the transferase block involves UDP-galactose formation from UTP and galactose 1-phosphate by uridine diphOSphate galactose perphosphorylase (Figure 1, reaction 6). Although this activity has been found in human liver (Isselbacher, 1957) the levels are low and do not increase with age (Abraham and Howell, 1969). Further, it is quite probable that the enzyme is not specific for galactose-l-phosphate and is in fact UDP- glucose perphosphorylase (Figure 1, reaction 5) (Knop and Hansen, 1970). Comparison of the Rat and Chick as Model Systems In order to delineate the biochemical mechanisms underlying disruption of normal cellular processes in galactosemia, most investigations have depended on changes induced in animals, most notably the rat and chick, by feeding high levels of galactose in their I diet. It must be remembered that since all the enzymes of the uridine nucleotide sugar phosphate pathway are present in these systems, the situation is not entirely analogous to a complete block at the transferase step. Success in inducing toxic symptoms depends upon over- loading the enzymes of the Leloir pathway. The major pathologic changes in rats fed 20 or 40 per cent of galactose in their diet include cataract formation, poor growth rate, and kidney damage charac- terized by amino aciduria (Craig and Maddock, 1953). Fetuses from pregnant rats fed high-galactose diet in addition demonstrate moderate fat infiltration of the liver (Spatz and Segal, 1965). Although brain abnor- malities are not usually observed (Handler, 1947), a combined ethanol-50 per cent galactose diet will evoke 10 neural degeneration, liver necrosis and death presumably due to inhibition of UDP galactose-4 epimerase (Tygstrup and Keiding, 1969). Quan-Ma and Wells (1965) demonstrated galactitol accumulation in liver, muscle, brain, kidney, and intestine of rats fed high-galactose diet. When 1 [1-14C] galactose or [1- 4C] glucose was administered to 4-day-old rats, no difference in the excretion 14 patterns of CO could be seen, suggesting that the 2 rat has a relatively good capacity for galactose oxi— dation via the Leloir pathway (H. Wells, Gordon and. Segal, 1970). Measurements of enzyme activities of the uridine nucleotide pathway in rat liver showed transferase levels to be the highest (Segal, 1971). The chick, on the other hand, develops severe neurotoxic symptoms including eliptiform convulsions, tremors, ataxia, and ultimately death when fed high levels of galactose (Dam, 1944; Rutter, Krichevsky, Scott and Hansen, 1953). Histological studies have revealed that the only significant lesion is in nervous tissue (Rigdon, Couch, Creger and Ferguson, 1963). Neurons, primarily in the basal ganglia, medulla, and occipital lobes were frequently found to be degenerated. Both galactose and galactitol accumulate in tissues, particularly in brain, kidney, and muscle 1 (H. Wells and Segal, 1969). Oxidation of [l- 4C] galac- 14 14 tose to CO2 occurred much slower than [1- C] glucose 11 oxidation and in tissue slice studies, brain and intestine demonstrated much lower galactose oxidation rates than kidney and liver (H. Wells, et al., 1970). In contrast 4C] galactonic acid did not with the rat, oxidation of [1 occur (Wells, §£_gl., 1970). Galactose-l—phosphate is known to accumulate in nervous tissue (Kozak and Wells, 1969), suggesting that galactose-l-phosphate uridyl trans- ferase is the rate limiting step in the pathway. However, evidence is conflicting. Transferase activity in the brains of female chicks was found to be one-half that of males and the lowest of the Leloir pathway enzymes (Mayes, Miller and Myers, 1970). Further, brain galactose- l-phosphate levels were twice those of male chicks, con- sistent with transferase enzyme as the rate-limiting step. 1 However, [1- or 2--4C] galactose oXidation studies with male and female chicks showed no difference in rates of 14CO2 evolution from the whole animals (H. Wells, gt_gl., 1970). Due to the high susceptibility to galactose neuro- toxicity, the chick has been adopted by several labora- tories as a model for the effects of galactose and its metabolites on the brains of galactosemics. Recent criticism of this model has suggested that hyperosmolality and not abnormal galactose metabolism causes the toxicity syndrome (Malone, Wells and Segal, 1971). Chicks given water containing 10 per cent galactose develop osmolalities 12 up to 470 milliosmols. However, when chicks are given a synthetic diet containing galactose (40 per cent by weight) osmolalities of only 335 are attained with normal levels at 305 (Knull and Wells, 1972). Knull and Wells also demonstrated that intraperitoneal injection of glucose into chicks fed the synthetic diet can reverse the gross symptoms of toxicity while raising the osmolality of the blood further, casting doubt on the role of hyperosmolality as a major factor in inducing neur0pathological symptoms in synthetic diet-fed animals. Possible RolesofGalactose, Galactitol and Galactose-l-Phosphate in the Etiology of Galactose Toxicity Syndrome Cataract Formation in the Lens.--Ga1actitol formation in the lens is considered to play the primary role in inducing cataracts by causing imbibition of water as a result of its poor diffusion properties (Kinoshita and Merola, 1964). In yitgg experiments with rat lens have shown that cataracts can be prevented by balancing the osmolality of the incubation medium with galactitol formation in the lens. Kinoshita and coworkers (1968) were also able to block water uptake and cataract formation with an inhibitor of aldose reductase, 3,3-tetramethylene-glutaric acid, for 3 days in an incubation medium containing galactose. The first 13 change occurring after water imbibition is a marked reduction of glutathione (Suppel, 1966) followed by decreases in amino acid levels (Kinoshita, Merola and Hayman, 1965), glycolytic enzymes and glycolytic rate (Suppel, 1967) within 2 days. Reduction in protein synthetic rate is also known to occur (Dische, §£_gl., 1957). Earlier attempts to implicate galactose-l— phosphate in cataract formation appear less likely as this metabolite accumulates late in the disorder. The finding that individuals with galactokinase deficiency also develop cataracts substantiates this conclusion (Gitzelmann, 1967). Aminoaciduria.--Ga1actose or galactose-l- phOSphate are thought to play a role in this disorder. Incubation of rat kidney cortex slices with galactose inhibits amino acid accumulation in kidney tubule cells (Thier, Fox, Rosenberg and Segal, 1964). Similar inhibition has been reported in rat intestinal mucosa incubated with galactose (Saunders and Isselbacher, 1965). The fact that patients observed so far with galactokinase deficiency do not demonstrate amino aciduria further implicates galactose-l-phosphate in the inhibition of renal amino acid transport (Gitzelmann, 1967). Biochemical Changes in the Brain.--Kozak and Wells (1969) observed reduction in levels of ATP by 15 to 14 20 per cent, phosphocreatine by 30 per cent, and energy charge by 10 per cent prior to onset of convulsive activity in chicks fed a high galactose diet. Further studies with radioactive tracers demonstrated a rapid turnover of galactose-l-phosphate while relatively little galactose was being metabolized (Kozak and Wells, 1971). This combined with the discovery of a phosphatase (Figure 1, reaction 4) with a low Km for galactose-l- phosphate and a Vmax several times greater than that for galactokinase or galactose-l-phosphate uridyl trans- ferase, led to the formulation of a novel mechanism shown below to explain the depression of ATP levels (Kozak and Wells, 1971). galactokinase Galactose + ATP ; Galactose-l-P + ADP phOSphatase Galactose-l-P + H20 % Galactose + Pi ATP + H20 e» ADP + Pi The result is a loss of ATP without further metabolism of galactose. Reduced cerebral glucose levels have also been implicated as a contributor to the depressed high energy phosphate reserves in chicks. Glucose and glycogen were found to be largely depleted during the later stages of the toxicity syndrome and high energy phosphates were rapidly utilized with little lactate formation during 15 ischemia (Granett, Kozak, McIntyre and Wells, 1972). Further, when the debilitated animals were injected intraperitoneally with glucose, there was a rapid though temporary recovery of the animals accompanied by an increase in brain glucose levels and a return of ATP and phosphocreatine to normal concentrations (Knull and Wells, unpublished results). However, it is doubtful that reduced glucose levels initiate the lowering of high energy phosphate levels. ATP is depressed by nine hours after galactose ingestion begins, while glucose levels have altered only slightly (Granett, gt_al., 1972). Impairment of glucose transport into the brain by high levels of serum galactose has been suggested as a cause for low brain glucose concentrations (Granett, g£_31., 1972). The total amount of [14C] glucose in the brain following intraperitoneal injection was considerably lower than that found in controls. In contrast, brain glucose levels are depressed to only a small extent and ATP levels are unaffected in rats fed high galactose diets (W. Wells, et_§l., 1969). This is presumably due to the greater capacity of rats to metabolize galactose via the sugar nucleotide phosphate pathway. However, other abnormalities have been observed in rat nervous tissue during galactose ingestion. Gabbay and Snider (1970) found decreased nerve conduction in sciatic nerve accompanied by accumulation of galactitol 16 and osmotic swelling, an effect similar to that in diabetic neuropathy in which sorbitol accumulates in nerve cells. Concentrations of selective amino acids are decreased in fetal rats when mothers are injected with large amounts of galactose (Carver, 1966). A decrease in the ability of seretonin to stimulate the contraction of stomach fundi in rat was shown to be a result of a deficiency in the seretonin receptors which are thought to be glycolipid in character (Woolley and Gommi, 1964). Galactose Toxicity in Cell Cultures-and Red Blood Cgll§.--The growth of human galactosemic fibroblasts is inhibited by galactose and cellular degeneration and death occurs by 72 hours. Electron micrographs have exhibited dilation of the endoplasmic reticulum during the incubation period (Miller, Gordon and Bench, 1968). ATP levels have been found to be reduced in galactosemic red blood cells during incubation with galactose (Penning- ton and Prankerd, 1958); however, this has not been con- firmed (Zipursky, §t_§1., 1965). E. 3911, with galactose- 1-phOSphate uridyl transferase deficiency show impaired growth rate in galactose containing media but galacto- kinase deficient mutants do not, suggesting galactose—l— phosphate as the toxic agent (Kurahashi K. and Wahba, 1958; Yarmolinsky, Wiesmeyer and Kalckar, 1959). 17 Subsequent studies by Sundararajan (1963) have shown galactose-l-phosphate interference with induction of glycerol kinase. Sidbury (1957) has proposed that galactose-1- phosphate inhibition of phosphoglucomutase is a major cause of galactose toxicity. The intermediate has been shown to accumulate in red blood cells (Sidbury, 1960) of galactosemics. However, inhibition has been shown only in_vitro and in the absence of cofactor glucose-1-6- diphosphate. REFERENCES Abraham H. and Howell R., J. Biol. Chem. 244, 545 (1969). Beutler E., Baluda M. C., Sturgeon P. and Day, R. W. Lancet 1, 353 (1965). Carver, M. J., Biochem. Biophys. Acta 130, 514 (1966). Craig J. and Maddock C., A.M.A. Arch. Path. 5;, 118 (1953). Cuatrecases P. and Segal S., J. Biol. Chem. 240, 2382 (1965). Cuatrecases P. and Segal S., Science 153, 549 (1966). Dam H., Proc. Soc. Exp. Biol. Med. _5, 57 (1944). Dische, Zelmenis G. and Youlous J., Amer. J. Opthal 44, 332 (1957). Donnell G. N., Koch R. and Bergren W. R. in Galactosemia (Edited by D. R. R. Hsia) p. 247. Charles C. Thomas, Springfield, Ill. (1969). Egan T. J. and Wells W. W., Amer. J. Dis. Child 111, 400 (1966). Gabbay K. H. and Snider J. J., Diabetes 19, 357 (1970). Gitzelmann R., Pediat. Res. 1, 14 (1967). Granett S. E., Kozak L. P., McIntyre J. P. and Wells W. W., (submitted for publication). Handler P., J. Nutrition 32! 221 (1947). Hayman S. and Kinoshita, J. H., J. Biol. Chem. 240, 877 (1965). Isselbacher K. J., Anderson E. P., Kurahashi K. and Kalckar H., Science 123, 635 (1956). 18 19 Isselbacher K. J., Science 126, 652 (1957). Isselbacher K. J. in The Metabolic Basis of Inherited Disease, 2nd Edition (Edited by Stanbury J. B., Wyngaarden J. B. and Fredrickson D. S.) p. 178. McGraw-Hill, New York (1966). Kinoshita J. H. and Merola L. 0., Invest. Ophthal 3, 577 (1964). Kinoshita J. H., Merola L. O. and Hayman S. J. Biol. Chem. 240, 310 (1965). Kinoshita J. H., Dvornik D., Kraml M. and Gabbay K. H., Biochem. Biophys. Acta 158, 472 (1968). Knop J. and Hansen R., J. Biol. Chem. 245, 2499 (1970). Knull H. R. and Wells W. W., Science (submitted for pub- lication), (1972). Komrower G. M. and Lee D. H., Arch. Dis. Child. 45, 367 (1970). Kozak L. P. and Wells W. W., Arch. Biochem. Biophys. 35, 371 (1969). Kozak L. P. and Wells W. W., J. Neurochem. 18, 2217 (1971). Kurahashi K. and Wahba A. J., Biochem. Biophys. Acta 30, 298 (1958). Leloir L. F., Arch. Biochem. Biophys. 33, 186 (1951). Malone J. J., Wells H. J. and Segal S., Science 174, 952 (1971). Mayes J. S., Miller L. R. and Myers F. K., Biochem. Biophys. Res. Comm. 39, 661 (1970). Merril C. R., Geier M. R. and Petricciani J. C., Nature 233, 398 (1971). Miller L. R., Gordan G. B. and Bench K. 6., Lab. Invest. 12, 428 (1968). Pennington J. S. and Prankerd T. A., Clin. Sci. 11, 385 (1958). Quan-Ma R. and Wells W. W., Biochem. Biophys. Res. Comm. ‘20, 486 (1965). 20 Quan-Ma R., Wells H. J., Wells W. W., Sherman F. E., and Egan T. J., Amer. J. Dis. Child. 112, 477 (1966). Rutter W. J., Krichevsky P., Scott H. M., and Hansen R. H., Poultry Sci. 32, 706 (1953). Sadler H. L., Inouye T. and Hsia D. R. R. in Galactosemia (Edited by D. R. R. Hsia) p. 127, Charles C. Thomas, Springfield, Ill. (1969). Saunders S. and Isselbacher K. J., Biochem. BiOphys. Acta 102, 397 (1965). Segal S. and Cuatrecases P., Amer. J. Med. 44, 340 (1968). Segal S. in The Metabolic Basis of Inherited Disease, 3rd Edition (Edited—by Stanburg J. B., Wyngaarden J. B. and Fredrickson D. S.) p. 174. McGraw- Hill, New York (1971). Sidbury J. 8., Am. J. Diseases Child. 94, 524 (1957). Sidbury J. B., Jr. in Molecular Genetics and_Human Disease (Edited by L. E. Gardner 77p. 61. Charles C. Thomas, Springfield, Ill. (1960). Spatz M. and Segal S., J. Pediat. 61, 438 (1965). Sundararajan T. A., Proc. Nat. Acad. Sci. 52, 463 (1963). Suppel T. 0., Invest. Ophthal. 5, 568 (1966). Suppel T. 0., Invest. Ophthal. 6, 59 (1967). Tedesco T. A. and Mellman W. J., Science 172, 727 (1971). Thier S., Fox M., Rosenberg L. and Segal S., Biochem. BiOphys. Acta 93, 106 (1964). Tygstrup N. and Keiding S., Nature 222, 181 (1969). Wells H. J. and Segal 8., FEBS Letters 5, 121 (1969). Wells, H. J., Gordon M. and Segal S., Biochem. BiOphys. Acta. 222, 327 (1970). Wells, W. W., McIntyre J. P., Schlichter D. J., Wacholtz M. C., and Spieker S. E., Annals of N.Y. Acad. Sci. 165, 599 (1969). Woolley D. W. and Gommi B. W., Proc. Nat. Acad. Sci. 52, 14 (1964). 21 Yarmolinsky M. B., Wiesmeyer H. and Kalckar H. M., Proc. Nat. Acad. Sci. 45, 1786 (1959). Zipursky A., Rowland M., Ford J. D., Haworth J. C., and Israels L. G., Pediatrics 35, 126 (1965). Figure 1. 22 Major metabolic pathways of D-galactose. 1. galactokinase (E.C. 2.7.1.6); 2. galactose 1-phosphate uridyltransferase (E.C. 2.7.7.12); 3. UDP glucose 4-epimerase (E.C. 5.1.3.2); 4. galactose 1-phosphate phosphatase; 5. UDP glucose pyrophosphorylase (E.C. 2.7.7.9); 6. UDP galactose pyrophos- phorylase (E.C. 2.7.7.10); 7. aldose reduc- tase (E.C. 1.1.1.21); 8. galactose dehydro- genase (E.C. 1.1.1. 48); 9. galactonic acid dehydrogenase; 10. UDP glucose pyrophospho- tase; ll. UDP galactose pyrophosphotase. 23 NADP’ NADPH NAD‘ NADHoH’ NDP‘ NADPH+ H’ «o H. Galactitol Galactose 0‘ Golactonlc Aci- ‘ Xylulose ATP T Pi ’6 UTP+ Glucose- l-P UDP 2° Galactose-l-P lucose (0 (NAD‘) 63 Glucose- l- P UDPGolactos 20 PP' :UIiJIP+Golactose-l-P UTP+ Golactose-l-P l Glycolysis CHAPTER II STUDIES ON AMINO ACID LEVELS AND PROTEIN METABOLISM IN THE BRAINS OF GALACTOSE- INTOXICATED CHICKS Abstract Levels of free amino acids, profiles of poly- ribosomes, and rates of protein synthesis and degradation were examined in the brains of chicks fed toxic levels of galactose. The content of a number of amino acids were altered; alanine and leucine were most strikingly depressed, whereas levels of aspartate were elevated. Polyribosomal profiles were unaltered. There appeared to be no detrimental effect on protein synthesis as 14C] leucine judged by in yizg incorporation of L-[U- and L-[guanidino-14C] arginine. Likewise, the half-lives of proteins, measured by the loss of L-[guanidino-14C] arginine, were similar in experimental and control groups. In contrast, initial rates of incorporation of [3H] glu- cosamine into glycoproteins were enhanced. The effect was greatest in the microsomal fraction and typically 50 per cent greater than controls. Levels of free 24 25 glucosamine and protein-bound hexosamine were essentially unaltered in the galactose-fed chicks. Introduction Galactose toxicity in the chick is characterized by ataxia, tremors, and severe seizures within 36-48 hours when animals are fed a diet containing 40 per cent (w/w) galactose (Dam, 1944; Rutter, Krichevsky, Scott and Hansen, 1953). Such neurological disturbances when induced by drugs, diet, or electroshock are associated with disruption of the control mechanisms for protein synthesis in the brain. Electroshock-induced seizures or high levels of phenylalanine are accompanied by dis- aggregation of polyribosomes in rats and mice (Vesco and Giuditta, 1968; MacInnes, McConkey and Schlesinger, 1970; Aoki and Siegel, 1970), and in studies of galactosemic fibroblasts, electron micrographs have revealed dilation of endoplasmic reticulum from polyribosomes (Miller, Gordon and Bensch, 1968). In rats aberrations in brain protein synthesis have been implicated in association with perturbation of levels of free amino acids induced by high levels of galactose (Carver, 1966) or of phynyl- alanine (Agrawal, Bone and Davison, 1970). Reduction in the rate of protein synthesis is accompanied by a depression of ATP levels in electroshocked brain slices (Lipmann, 1970). Furthermore, the rate dependence of histidyl tRNA synthetase in Salmonella 26 tymphimurium is known to be sensitive to energy charge in yitg2_(3renner, Delorenzo and Ames, 1970). These latter observations are of particular interest in light of the reported depression of brain ATP, creatine phos- phate, and energy charge in the galactose-intoxicated chick (Kozak and Wells, 1969). Accordingly, the present study was undertaken to investigate the effects of galactose toxicity in the chick on polyribosomal aggregation, pool sizes of free amino acids, and resultant effects on protein synthesis and degradation in the brain. Protein turnover, in subcellular fractions, as well as in total brain, was studied in yi!2_with [14C] leucine or [14C]arginine, and one protein subclass (glyc0protein) was examined with [3nglucosamine. Materials and Methods Animals and Materials Day-old Leghorn cockerels, purchased from Cobbs, Inc. (Goshen, Ind.) were housed in a brooder at 32°C. Animals were placed on a synthetic diet described by Rutter §£_§1, (1953), with 40 per cent (w/w) of the diet replaced with D-galactose (General Biochemicals, Inc., Chagrin Falls, Ohio). L-[U-14C]Leucine was obtained from either New England Nuclear (Boston, Mass.; 23'6 mCi/mmol) or Amersham Searle (Chicago, 111.; 344 mCi/mmol; 27 L-[guanidino-14C] arginine was from New England Nuclear (4.58 mCi/mmol) or from Amersham/Searle (25 mCi/mmol), and D-[6-3nglucosamine (3.6 Ci/mmol) was from New England Nuclear. Glucose 6-phosphate dehydrogenase (D—glucose 6-phosphate: NADP oxidoreductase; EC 1.1.1.49) and pyruvate kinase (ATPzpyruvate phosphotransferase; EC 2.7.1.40) were purchased from Boehringer Mannheim (New York, N.Y.), and hexokinase Type C-300 (ATPzD-hexose 6-phosphotransferase; EC 2.7.1.1), and lactate dehydro- genase Type III (L-lactate: NAD oxidoreductase; EC 1.1.1.27), were purchased from Sigma Chemical Co. (St. Louis, Mo.). In Vivo Tracer Studies Individual control and experimental chicks were injected intracerebrally with 10-20 ul of labelled pre- cursor in 0.15 M-NaCl solutions. Animals were decapi- tated at appropriate times, and whole brains were removed and placed in ice or ice-cold buffer (0.01 M-tris, pH 7.0, in 0.25 M-sucrose) (see Legends to figures for details). Brains were weighed and homogenized in 5 vol. of the same buffer in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 850 g for 10 min. to obtain a crude nuclear fraction which was further purified for nuclei according to the method of Casola and Agranoff (1968). For a crude mitochondrial fraction, the 850 g 28 supernatant fraction was then sedimented at 1,000 g for 20 min. Microsomal and soluble protein fractions were separated by centrifuging the 11,000 g supernatant fraction at 100,000 g for 90 min. Preparation of Samples for Counting Protein was treated according to the method (Method A) described by Lim and Agranoff (1966) for counting. The powdered tissue was weighed on an analytical balance, dissolved in 0.5 m1 of hyamine hydroxide for 3 min. at 80°C, and then mixed with 10 m1 of toluene-based scintillation fluid (2,5- diphenyloxazole, 5 g; 1,4-bis-[2-(5-phenyloxazoly1)]- benzene, 0.3 g; toluene to 1 litre). Radioactivity was then counted in a Beckman c.p.m. 100 liquid scintillation spectrometer as c.p.m. and corrected to d.p.m. by means of an external standard ratio and quenching curve for either 14C or 3H, as required. Efficiency for full channel l4C c.p.m. was 95 per cent. In the glycoprotein experiments, the method of Robinson, Molnar and Winzler (1964) was followed for isolation of the protein fraction and subsequent counting of radioactivity; the procedure was modified slightly by adding 4 per cent (w/v) Cabosil to the scintillation fluid composed of 10 g of 2,5- diphenyloxazole, 100 g of naphthalene and 1 litre of dioxane. Protein concentrations were measured by the 29 method described by Lowry, Rosebrough, Farr and Randall (1951) after treatment with 0.2 M-sodium hydroxide treatment for 30 min. at 90°C. Determination of the Specific Radioactivity of Free {135] Leucine The TCA-soluble supernatant fraction was extracted six times with diethyl ether to remove TCA and then treated with Dowex 50W-X8 [H+] (50-100 mesh) to separate amino acids from sucrose. Samples were reduced in volume with a flash evaporator, spotted on Whatman 3 MM paper, and chromatographed overnight with butanol-acetic acid- water (4:1:5, by vol.). The area on the chromatogram corresponding to leucine, isoleucine, and phenylalanine was eluted with water and diluted to a known volume. A portion of this sample was counted in the dioxane-based scintillation fluid. Isoleucine and phenylalanine, being essential amino acids, are unlabelled and do not interfere with the radioactive determinations. Another portion was treated according to the method of Gehrke and Stalling (1967) to convert the amino acids to the corresponding N-trifluoroacetyl-n-butyl ester. The derivatives were analyzed quantitatively with proline as an internal standard on a Tabsorb column with a Hewlett-Packard Model 402 gas chromatograph. 30 Preparation of Tissue for Free Amino Acid_Quantification Chicks fed respective diets for 44 hours were decapitated and the brains were removed within 15 seconds and frozen in liquid N Tissue was powdered at dry ice 2. temperature and stored at -90°C until needed. Amino acids were extracted and prepared for analysis by a modification of the procedure described by Levi, Kandera and Lajtha (1967). Tissue was treated with 5 vol. of 0.6 M-HClO4, centrifuged to remove the residue, and the HClO4-solub1e fraction was neutralized with 1.2 M-KOH; the KClO4 was removed by centrifugation at 0°C. The extract was treated with 50 umol of Na 803 and 2 10 umol of cysteine per g of original tissue to oxidize glutathione and remove it from the glycine-alanine region of the chromatogram. Samples corresponding to either 4 or 40 mg of original tissue were then applied to a one-column amino acid analyzer using the gradient system of Piez and Morris (1960). Polyribosomal Profiles Polyribosomes were isolated from chick brain by the method described by Earl and Morgan (1968) in a homogenizing medium consisting of 0.02 M-tris (pH 7.6); 0.001 M-EDTA; 0.1 M-KCl; 0.01 M- or 0.004 M-magnesium acetate; and 0.2 M-sucrose. Polyribosome pellets could be stored at -90°C for two weeks without alteration 31 in polyribosomal profiles. Polyribosomal pellets isolated from pools of eight chick brains were lightly diapersed in 0.75 ml of resuspension buffer with a loose- fitting Potter-Elvehjem homogenizer, and corrected to approximately the same concentrations (A260 nm/mg ribo- somes = 11.3). Portions were placed on 15-30 per cent (w/v) linear sucrose gradients containing the buffer already described with 4 mM-magnesium acetate, and tubes were centrifuged at 25,000 rev./min for 3.5 hours in a 25-1 SW rotor in a Beckman Model L2 centrifuge. Tubes were punctured at the bottom, and the gradient was pumped through a flow cell (0.5 mm) of a Gilford Model 2000 spectrophotometer at 260 nm. Determination of Protein- Bound Hexosamine Dried protein fractions were hydrolysed for 3 hours in 4 M-HCl (2 ml/mg) at 100°C. Hexosamine was determined by a modification of the Elson and Morgan method for amino sugars (Boas, 1953). Estimation of the Specific Radioactivity of Free i3HI Glucosamine Groups of eight chicks were injected intracere- brally with l uCi of [3H] glucosamine 44 h after being placed on control and experimental diets and after 10 or 20 min. were decapitated into liquid N2. Brains were chipped out and powdered in a dry ice-chilled pan 32 and mortar and pestle at a temperature of -50°C. We have observed that values for labile intermediates obtained by this procedure are indistinguishable from those determined for brains chipped out of skulls in a walk-in freezer at -20°C. The following operations were all carried out at 4°C. Portions of the powdered tissue were homogenized in a Potter-Elvehjem homogenizer with 5 vol. of 10 per cent (w/v) TCA. The TCA-insoluble material was sedimented by centrifugation at 12,000 g for 10 minutes and the TCA was extracted from the super- natant fluid with five successive treatments with an equal volume of diethyl ether. The extract was adjusted to pH 7 and passed through a Dowex 50 X 8 (NH4+ form) (200-400 mesh) column (7 cm X 0.9 cm dia.) according to the method described by Thompson, Morris and Gering (1959). After washing the column with 50 m1 of H O, the 2 4OH. The NH4OH was removed by lyophilization and the residue was amino compounds were eluted with 60 m1 of 2 M-NH taken to a known volume with H20. Essentially all of the radioactivity in the NH4OH eluate co-chromatographed with standard glucosamine on Whatman 3 MM paper in a butanol-pyridine-H20 (6:4:3, by vol.) solvent system. Glucosamine was quantified by a modification of the fluorometric assays for glucose and ATP described by Lowry, Passonneau, Hasselberger and Schulz (1964). Glu— cosamine was converted to glucosamine 6-phosphate with 33 hexokinase (EC 2.7.1.1) and the ADP formed was quantified by coupling with pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27), and observing the oxidation of NADH. The assay mixture contained 0.1 M-tris HCl (pH 7.5); 4 mM-MgCl 80 mM-KCl; 0.25 mM-ATP; 0.125 mM 2. phospho-gpglfpyruvate; 1.4 UM-NADH; 1 01 of lactate dehydrogenase; 3 ul of pyruvate kinase; 1 ul of hexo- kinase; and H20 and extract to a final volume of 2.0 ml. Samples were corrected for any residual glucose contami- nation by the enzymic method described by Lowry gg_§l. (1964). Glucosamine and glucosamine 6-phosphate did not intere with the glucose 6-phOSphate dehydrogenase assay. In order to test the isolation procedure for glucosamine, 12.6 nmol of standard glucosamine, as judged by the assay, were added to a TCA homogenate of 2 g of brain tissue obtained as described above. Triplicate samples equal to 0.5 g of original tissue, with and without the added glucosamine, were then carried through the isolation procedure described above. Added glucosamine was deter- mined by the fluorometric assay on samples equivalent + to 0.1 g of original tissue and found to be 0.67 - 0.06 nmol or an average recovery of 106 per cent. 34 Results [14C]Leucine Incorporation in Vivo into Brain Protein A number of amino acid levels were altered in chicks fed galactose (Table 1); however, glutamine, glutamate, aspartate, leucine, and alanine appeared to be most markedly affected. The severity of decrease in the latter two (25 and 40 per cent, respectively), compounded with their already low levels, suggested possible inhibitory effects on rates of protein synthesis at the level of their respective tRNA charging enzymes. To investigate this possibility, as well as the effect of lowered ATP levels on protein synthesis in the 4C]1eucine into galactose-fed chick, incorporation of [1 protein ip_!izg was studied over a 20 minute period. As shown in Table 2, measurements based on d.p.m./mg of protein alone are deceptive. The rate of incorporation of [14 C]1eucine is based not only on the rate of poly- peptide formation but also on the concentration and, hence, the initial specific radioactivity of free leucine. In the case of the galactose-fed chick, the lower level of free leucine resulted in a higher initial specific radioactivity and was reflected in the greater incor- poration of radioactivity into protein. After corrections were made for the specific radioactivity of the precursor, virtually no difference in incorporation of leucine into 35 protein was detected. When protein samples from this eXperiment were subjected to 72 hours hydrolysis at 110°C in 6 M-HCl, essentially all radioactive material in the hydrolysate cochromatographed on paper with leucine in the butanol-acetic acid-water solvent system. Polyribosomal Profiles and Rates of Synthesis of Nascent Poly- peptide Chains Since the above experiment was based on analysis 14C] at only one time, the experiment was repeated with [U- 1eucine, measuring only the rate of synthesis of the nascent polypeptide on the polyribosomes. The incorpor- ation of [U-14C]leucine into polypeptide (d.p.m./mg ribosome) is approximately 25 per cent faster in the galactose-fed animals, (Figure 2), but if corrected for the elevated specific radioactivity of leucine, the incorporation curves would be identical. Polyribosomes isolated in this same experiment were centrifuged on sucrose density gradients and sedi- mentation rates detected with a flow cell as outlined in Materials and Methods. Representative profiles from a number of runs are presented in Figure 3. The ratios of monoribosomes to polyribosomes, calculated by inte- grating separately the areas of the monoribosomal peak and polyribosomal peaks of three separate experiments + 1- were 0.100 - 0.010 for control and 0.099 0.012 for 36 galactose-fed chicks. Yields were typically 0.50 mg of ribosomes/g of brain from both control and experimental groups. In Vivo Catabolism of Brain Protein Studying rates of degradation of proteins is complicated in these experiments by the relatively short life of the chicks fed galactose in comparison to the long half-lives of most brain protein. ATP levels fall and galactose 1-phosphate levels rise after 9 hours of galactose feeding (Kozak and Wells, 1971), but tremors or convulsions are not seen until 30-36 hours after initiation of the diet, with death of the chicks ensuing by 48-54 hours. To partially circumvent this difficulty, chicks were injected intracranially with L-[guanidino-14C] arginine 2 hours before placement on the respective diets. Figure 4 illustrates the degradative rates of protein over the 48-hour period. The average half-life of total protein over the 2-day period was approximately 36 hours for both galactose- and control-fed chicks. Likewise, no significant difference in the decay curves of the sub- cellular fractions could be detected; apparent half-lives of degradation were 36 hours for the soluble fraction and 60 hours for the microsomal fraction. The biphasic char- acter of the curve for the crude mitochondrial fraction 37 precluded half-life determinations; however, it did not appear to reflect a recycling of labelled arginine. The radioactivity in the TCA-soluble fraction 24 hours after injection was typically less than 500 d.p.m./g of brain, and as estimated by paper chromatography in butanol-acetic acid-water (4:1:5, by vol.), was largely [14C1urea. Biosynthesis of Glycoprotein Advantage was taken of the previously observed equal rates of protein synthesis from [14C]leucine in experimental and control animals by using both [6-3H] glucosamine and L-[guanidino-14C1arginine as glycoprotein precursors. The ratio of 3H to 14 C indicated the rate of synthesis of the carbohydrate moiety of glycoprotein relative to that for total protein synthesis. Arginine was also chosen because of its unaltered levels in brains of galactose chicks (Table 1). [3H] Glucosamine was incorporated at a significantly enhanced rate into microsomal and crude mitochondrial protein fractions in the galactose-fed chicks (Figure 5). The observed increase in incorporation from [3H]glucosamine was most obvious in the glyc0protein-rich microsomal fraction, whether expressed on the basis of per mg of protein or relative isotOpe ratio (Figure SB). Levels of free glucosamine, as determined by the fluorometric assay, were similar (1.9 t 0.1 nmol/g of tissue for controls, 2.2 i 0.1 nmol/g of tissue for 38 galactose-fed), an observation suggesting that a precursor, specific radioactivity effect as seen with the [14C]1eucine studies did not exist. To further investigate this pos- sibility , the specific radioactivities of free glucosamine were measured in a separate experiment (described in Methods). Specific radioactivities at 10 and 20 minutes after injection were 190,000 d.p.m./nmol, and 95,000 d.p.m./nmol, respectively, for controls and 30,000 d.p.m./nmol and 20,000 d.p.m./nmol for galactose-fed animals (range of error, approximately 20 per cent). These values were indicative of a faster rate of utili- zation of free glucosamine which was consistent with the enhanced incorporation of tritium into glycoprotein. Preliminary measurements demonstrated that 80 per cent of the tritium in the total protein fraction at 30 minutes was associated with [3H]hexosamine. Also, hexosamine levels in the total protein fraction were identical and typically 8.2 i 0.8 and 8.5 i 1.3 ug/mg of protein for control and galactose-fed animals, respectively. Discussion In general, our amino acid analyses are in good agreement with those reported by Levi eE_gl. (1967) for hen brain. The slight variations observed may reflect the developmental stage of the animal, an effect seen in other instances (Agrawal, Davis and Himwich, 1969). 39 Earlier studies have shown that high levels of galactose alter the levels of brain amino acids. Carver (1966) demonstrated a decrease in concentration of a number of amino acids in fetal rat brain following injection of galactose into the mother. The alterations of free amino aCid levels observed in our studies do not correlate well with those reported for fetal rat brain except for the similar decreases in alanine and leucine. A variety of factors, including mode of introduction of D-galactose (diet versus injection), duration of exposure to high levels of galactose (2 days versus 1 h), or differing susceptibility of the two species to galactose intoxi- cation, may contribute to the dissimilarities. It is difficult to ascribe alterations in levels of amino acids seen under galactose feeding to any one factor. The altered concentrations of amino acids associated with the glycolytic pathway and the tricarbo- xylic acid cycle (alanine, aspartate, glutamate) may reflect changes in metabolic flux rates of these path- ways in response to increased energy requirements. Alanine levels, in particular, may be depressed as a result of a greater diversion of pyruvate into the tri- carboxylic acid cycle. Restricted energy reserves may cause selective impairment of cellular membrane transport systems for different amino acids. Battistin, Grynbaum and Lajtha (1969) have demonstrated in brain slices that 40 the sensitivity to a decrease in the energy supply was not identical for all amino acids and varied with the type of inhibitor used to restrict energy reserves. Whether galactose directly competes with certain amino acids for transport in brain has not been adequately assessed to our knowledge, although evidence for a common carrier of galactose and selective amino acids in intestine appears to be conflicting (Munck, 1968; Saunders and Isselbacher, 1965). The alteration in amino acid levels and the reduction of ATP content appear to have had no effect on turnover of brain proteins in chicks fed galactose for 48 hours. This conclusion was demonstrated by the identi- cal time course for the labelling of protein in subcellular fractions with [14C]arginine and [14C]leucine, the [14C] leucine incorporation into peptidyl tRNA, the similarity of the polyribosomal profiles, and equal half-lives of protein in the control and experimental animals. One difficulty in correlating altered levels of free amino acids with_a possible regulatory role in rates of protein synthesis is the uncertainty of the effective concentration of amino acids in different cytoplasmic locations and in different cell types. Apparently, leucine and alanine levels localized at the sites of protein synthesis in the cytoplasm, mitochondria and nucleus are sufficiently higher than the Km of their respective tRNA synthetases 41 and, therefore, exhibit little control function. Like- wise, the lack of observed control of rates of protein synthesis by the high-energy phosphate levels may reflect unaltered levels of ATP at protein synthetic sites or insensitivity of the energy-requiring reactions 13 vivo to moderate reductions in the adenine energy charge. The half-lives calculated for degradation of protein were substantially lower than those reported in adult rat brain by Piha, Cuénod and Waelsch (1966), and Lajtha (1959). Several factors, including age of animals, type of radioactive amino acid used, and time course over which the degradation rates were followed, may all contribute to the faster decay rates which we observed. That protein synthesis slows down in mammalian brain during latter stages of development has been well substantiated (Johnson and Luttges, 1966), and may be accompanied by a deceleration of degradation rates. Arias, Doyle and Schimke (1969) have examined the effect of different amino acid tracers on measured half-lives of protein in liver. The recycling of radio- activity, more substantial with other amino acids, was minimized with L-[guanidino-14C]arginine. Finally, our studies were carried out over a relatively short time period (48 h) and, thus, would largely reflect the degradation rates of the proteins turning over most rapidly. 42 The biphasic nature of the curve for the crude mitochondrial fraction (Figure 3) may reflect the two processes of degradation of protein in mitochondria and of incorporation of labelled protein into myelin and nerve endings by ax0plasmic flow--both present in this fraction. Reports by Piha gg_gl. (1966) and Barondes (1968) suggest that both of these processes function simultaneously. One source of error in our protein degradation experiments is the non-steady state level of protein metabolism; i.e. there is a net increase in protein with time in the growing chick, with the effect of decreasing the observed half-life. However, brain wet weight increases less than 10 per cent over the 2—day period, and because of the well-developed nature of the young chick brain, one would not eXpect more than parallel increases of 10 per cent in protein. Most interesting is the accelerated rate of incorporation of [3nglucosamine into protein fractions in the galactose-intoxicated chicks. The much lower values for specific radioactivity of free glucosamine in the galactose-fed chick in comparison to those in control animals are indicative of a much faster rate of turnover of the free glucosamine pool, presumably because of increased requirements for glycoprotein and/or glyco- lipid biosynthesis. The increased rate of incorporation 43 may reflect elevated rates of synthesis of glycoproteins or only their carbohydrate moieties. Since protein- bound levels of hexosamine were equal in the two dietary groups, increased rates of synthesis would have to be accompanied by increased rates of degradation. Whether the apoprotein is being synthesized at a greater rate 14C] cannot be ascertained from present experiments. [ Arginine was incorporated at the same or slightly faster rates into protein of each subcellular fraction studied in the galactose-fed chicks. However, these rates are an average of individual synthetic rates of all types of protein. The increased incorporation of tritium into glycoprotein could also be explained by either an alteration in the regulatory control of the enzymes converting glucosamine into other carbohydrate pre- cursors of glycoproteins or an elevation of the Specific radioactivity of the nucleotide N-acetyl hexosamine intermediates as a consequence of depression of their concentrations. These questions, together with possible parallel effects in the glycolipids, are subjects of further investigation in our laboratory. Acknowledgments The advice and assistance of Dr. H. Knull in the glycoprotein studies are gratefully acknowledged. We also wish to thank Dr. D. Robertson and Mrs. Diana Ersfeld 44 for their technical assistance in the amino acid analyses and Drs. R. Slabaugh and A. Morris for suggestions in the analysis of polyribosomes. REFERENCE S Agrawal H. C., Bone A. H. and Davison A. N., Biochem. J. 117, 325 (1970). Agrawal H. C., Davis J. M. and Himwich W. A. in Handbook of Neurochemistry (Edited by A. Lajtha), Vol. 1, p. 34. Plenum Press, New York (1969). Aoki K. and Siegel F., Science, N.Y. 168, 129 (1970). Arias I. M., Doyle D. and Schimke R. T., Biol. Chem. 244, 3303 (1969). Barondes S. H., J. Neurochem. 15, 343 (1968). Battistin L., Grynbaum A. and Lajtha A., Brain Res. 55, 187 (1969). Boas N. F., J. Biol. Chem. 204, 553 (1953). Brenner M., Delorenzo F. and Ames B. N., J. Biol. Chem. 245, 450 (1970). Carver M. J., Biochim. Biophys. Acta 130, 514 (1966). Casola L. and Agranoff B. W., Brain Res. 55, 227 (1968). Dam H., Proc. Soc. Exp. Biol. Med. 55, 57 (1944). Earl D. C. N. and Morgan H. E., Archs BiOChem. Biophys. 128, 460 (1968). Folbergrova J., Passonneau J. V., Lowry O. H., and Schulz D. W., J. Neurochem. 55, 191 (1969). Gehrke C. W. and Stalling D. L., Separation Sci. 2, 101 (1967). Johnson T. C. and Luttges M. W., J. Neurochem. 55, 545 (1966). 45 46 Kozak L. P. and Wells W. W., Archs Biochem. Biophys. 135, 371 (1969). Kozak L. P. and Wells W. W., J. Neurochem., in press (1971). Lajtha A., J. Neurochem. 5, 358 (1959). Levi G., Kandera J. and Lajtha A., Archs Biochem. Biophys. 119, 303 (1967). Lim R. and Agranoff B. W., J. Neurochem. 15, 431 (1969). Lipmann F. in Protein Metabolism of the Nervous System (Edited by A. Lajtha), p. 305. Plenum Press, New York (1970). Lowry O. H., Passonneau J. V., Hasselberger F. X. and Schulz D. W., J. Biol. Chem. 239, 18 (1964). Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J., J. Biol. Chem. 193, 265 (1951). MacInnes J. W., McConkey E. H. and Schlesinger K., J. Neurochem. 11, 457 (1970). Miller L. R., Gordon G. B. and Bensch K. G., Lab. Invest. 15, 428 (1968). Munck B. G., Biochim. BiOphys. Acta 156, 192 (1968). Piez K. A. and Morris L. A., Analyt. Biochem. 1, 187 (1960). Piha R. S., Cuénod M. and Waelsch H., J. Biol. Chem. 241, 2397 (1966). Robinson G. B., Molnar J. and Winzler R. J., J. Biol. Chem. 239, 1134 (1964). Rutter W. J., Krichevsky P., Scott H. M. and Hansen R. G., Poultry Sci. 55, 706 (1953). Saunders S. J. and Isselbacher K. J., Nature, Lond. 2 6, 700 (1965). Thompson J. F., Morris C. J. and Gering R. F., Analyt. Chem. 51, 1028 (1959). Vesco C. and Giuditta A., J. Neurochem. 15, 81 (1968). TABLE 1. 47 Analysis of Free Amino Acids and Ammonia in the Brains of Chicks Fed Control or Galactose-Containing Diets. Control Galactose Compounds (nmol/g wet wt.) Aspartate 2.310 c 0.071 3.127 i 0.091* Glutamate 7.642 i 0.148 6.912 1 0.1231 Glycine 1.059 t 0.111 1.008 i 0.010 Alanine 0.273 a 0.003 0.172 : 0.005+ Valinel 0.064 i 0.004 0.058 i 0.004 Methionine 0.052 1 0.005 0.050 1 0.002 Isoleucine 0.0197 0.002 0.0181 0.0017 Leucine 0.05l : 0.001 0.038 5 0.0031 Tyrosine 0.042 1 0.002 0.034 1 0.003§ phenylalanine1 0.061 i 0.003 0.071 i 0.005 y-Aminobutyrate 2.010 1 0.088 1.759 t 0.063 Lysine 0.553 i 0.023 0.471 1 0.0115 Histidine 0.222 i 0.004 0.194 1 0.0095 Tryptophan 0.021 t 0.002 0.020 r 0.005 Arginine 0.138 t 0.003 0.133 t 0.005 Ammonia2 0.526 i 0.103 0.630 r 0.087 Glutamate2 8.000 r 0.117 6.085 1 0.388+ Glutamine3 5.913 t 0.107 5.130 a 0.062* * 1 p < 0.01; +9 < 0.005; 19 < 0.025; 59 < 0.05; valine chromatographs with galactosamine and phenylalanine with B-alanine;2Determined fluorometrically (Folbergrova, Passonneau, Lowry-and Schulz, metrically as glutamate by difference before and after hydrolysis for 2 h at 100°C with 2 M-H Each value represents an average (s.d.) of 1969);3 Determined fluro- three separate determinations on a pool of 12 brains from each experimental group. analyser because of overlap. Serine, threonine, glutamine, and asparagine were not quantified by the amino acid 48 TABLE 2.--Incorporation of L-[U-14C1Leucine into Brain Subcellular Fractions. d.p.m./mg of Protein d.p.m./unmole Fraction Diet cg°§£§téig of free Leucine Supernatant Control 2720 t 130 .6200 i 250 Galactose 3500 i 380 ”.7030 i 600 Microsomes Control 4930 i 800 13,000.: 1250 Galactose 6750 i 350 - 13,250 i 400 Crude Control 4760 i 630 12,610 i 900 Mitochondrial Galactose 6370 i 450 12,800 i 600 Nuclear Control 2120 t 300 5625 i 400 Galactose 2450 i 450 5000 i 750 Male, day-old chicks (eight/group) were fed the respective diets for 44 h. They were injected intra- cranially with 20 ul (1 uCi) of L-[U-14C]leucine (2 uCi/ug) and killed 20 minutes later. Each value represents an average of determinations from two pools of four brains (i range). Figure 2. 49 Incorporation of L-[U—14C]leucine into pep— tidyl tRNA on the polyribosomes. Control and galactose-fed chicks fed their respective diets for 44 h were injected intracranially with 0.5uCi of L-[U-14C]leucine. Animals were killed at times indicated in the Figure (eight per experimental group per time period) and polyribosomes were isolated as described in the text section on Methods. After diluting the polyribosomes to equal concentrations, triplicate aliquots were prepared for counting of radioactivity. Ribosomes and protein were precipitated with 10% (w/v) TCA at 0°C for 30 minutes, centri- fuged, and then heated at 90°C for 20 minutes with 10% (w/v) TCA. The precipitate was col- lected on a glass filter (Whatman GF/C, 2.4 cm), washed with 5% (w/v) TCA, dried, and counted with the glass filter in toluene-based scintillation fluid. Standard deviations are denoted by vertical bars. 8000 6000 4000 C PM/ mg ribosomes 2000 50 0 Control 0 Galactose Time, (mins) 51 .cofluoom moocumz mom .maflmuoo Hmucoafiuomxm Mom .czonm mum mucoafluwmxo Hmowmmu 03¢ Eoum moaflmoum HmEOmOQHHSHom .m musmfim 52 25223.5 yo Ectom E9.“— 02.2.6.5 m. N. N. m O on vm - fi 1 omo~oo_00 l - .8250 mob 9.0 ¢N.O N00 0930 .0 53 .OIIIIIO .omouooamw «QIIIIIO .Houucou .mcofiumcHEuouoo oamfluHoE Mom ucoo Dom oa toooooxo maouou mcoflumfi>oo onmocoum .Ammmav mmocmumd one awn mo 0 conuoz an mua>fluomowomu mo mcwucsoo MOM ooummoum new oumcomoaon mosuo on» Eonm coxmu mums monEmw HE A o>wm .cwououm annoy Dom .moosuoz cw oocwauso mm doom some so come ouo3 mcofiumcfiEuoumo oumoflamflua .ousmfim on» GM ooumofiocfi moEHu um ooaawx ouo3 msoum Hmucoefiuooxo some Eoum mxoflno o>Ho3B .uouma muson N muowo mcwocommouuoo on» so ooomam one ocwcflmum"OvalocwowcoomHlA mo HO: m.o nufi3 maaoasmuomupcw oouooncfl ouo3 meHno UHOISBQ .ocacfimmm ”Ovalocwowcmsmglq nufi3 ooumuumoaafl mm cofiumomumoo cflmuoum mo moumu o>w> cH .v muomflm 54 .36... .6 5:25.. .336 55.25... we on ¢~ ~— 0 we on cm a 1 d d u u d 5221 3.63525 umord/ 0w w d o q q q q £261 3E8oeo§ .00m 529:. 0323 CON 00v uuolq tub / WdO ,_ I umord 601/ w d o 55 .mnen Heofiuuo> an ooueowocw mue mcowuew>oo oueoceum .Ollnlo .omouoeaew “.IIIIC .Houucoo .cowuoeum measaaoonom mcflocommouuoo on» cw oowuom 08w» noem ue ofiueu Uva\mm on» on muomou m ca Domes mos .mao>wuommmou .mcofluoenm Hewuoconooufie mosno oce Hesomouowa .oHnsHOm on» mo coHuoeum caououm on» ouca muw>fluoe0woeu mo cowpeuomuoocw ucomoumon 0 one .m .d .ucoEHummxm ocoomm e CH omcweuno mumz mpaommu HeowucooH .musmflm on» CH ooueowocfl moEHu De Aoofluom oEHu noeo How mooum Heucwafiummxo Mom NHV ooaawx one muofio mo coauefluflcw Houme a vv memouomfi o>fluoommou en» mo soeo won H £DH3 maaewceuoeuucw ooaoomcw ouoz meREO .mxofiso oomuaouucoo one nomouoeaem mo mcofiuoeum camuoum neaoaaoooom ucouommwo on» ODGH ocflcflmue”Deanocaoflcesmglq oce ocfleemoosHmHmmlmalo mo cofiueuomuoocH .m ouomflm 56 18.8%. 5.6 2.5 as: 21.. 5.82682 8.. 55852.: ummd bun/wag CHAPTER III BIOSYNTHESIS OF GLYCOPROTEIN, GANGLIOSIDES AND MUCOPOLYSACCHARIDE IN BRAINS OF CHICKS FED D-GALACTOSE Introduction Glycoproteins, known to be abundant in neuronal and synaptic membranes, are thought to play a role in intercellular recognition by neurons (Gesner and Ginsberg, 1964; Crandall and Brock, 1968; Barondes, 1970) and hence, in the establishment of neural pathways. Abnormal glyco- protein metabolism, particularly at the synapse, might be expected to have adverse effects on normal brain develop- ment. Previous studies have shown an enhanced incorpor- ation rate of [3H] glucosamine into the glycoprotein fraction of brains of chicks suffering from galactose toxicity while free and protein bound levels of glucosamine were essentially unaltered (Chapter II), suggesting the possibility of a faster turnover rate of the carbohydrate moieties. There are, however, disadvantages to using glucosamine as a precursor for glycoprotein synthesis because of the cell's ability to metabolize glucosamine 57 58 by more than one pathway and the existence of several intermediates between glucosamine and glycoprotein. In the latter case, a change in the pool size of any one of the intermediates could affect the incorporation rate of the labelled precursor into glycoprotein without an actual change in true rate occurring. Glucosamine is also a precursor for ganglioside and mucopolysaccharide synthesis; hence the increased incorporation of radioactive tracer into glyc0protein may be secondary to an increased biosynthesis of either one of these classes of molecules. To better characterize ig_zivg glycoprotein syn- thesis rate in chicks fed galactose, use has been made of other radioactively labelled precursors of glycomacromole- cules. [3H] Mannosamine, a precursor to N-acetylneuraminic acid, demonstrated an enhanced incorporation rate into both glycoproteins and gangliosides while NaBSSO4 utili- zation for mucopolysaccharide biosynthesis appeared to be normal. In addition, [an] glucosamine incorporation into gangliosides and mucopolysaccharide was measured. Materials and Methods Animals and Materials Day-old cockrels were purchased from Klagers (Manchester, Michigan) and housed in a brooder. Animals were placed on synthetic diets described by Rutter, Krichevsky, Scott, and Hansen (1953) with 40 per cent 59 galactose (w/w) in place of a corresponding quantity of cerelose in the experimental group. Fresh water was provided, ad libitum. D-[6-3H] glucosamine (3.6 Ci/mmole); 35 Na SO4 (496 mCi/mmole) and D-[6-3H] mannosamine (3 Ci/ 2 mmole) were all purchased from New England Nuclear (Boston, Massachusetts). All other chemicals were of reagent grade. In Vivo Tracer Studies Animals were fed their respective diets for 44 to 48 hours. At the end of this period, tracers were injected intracrainally in 20 ul of 0.154 N NaCl. In the amino sugar experiments, 1 uCi was given to each chick while 2 35504 was injected per animal to the muco- S uCi of Na polysaccharide study. Twelve chicks were used per time period per experimental group for the glucosamine experi- ment while groups of 10 animals per time period were used in the rest of the studies. At the end of the appropriate time period (see legends to figures for details) the chicks were sacrificed and brains removed, pooled and placed in ice cold water. Tissue was drained of excess liquid and then homogenized in 6 volumes of water in a Potter-Elvehjem homogenizer. When subcellular fractions were to be prepared, brains were placed in 0.32 M Sucrose, with 1 mM Tris, pH 7.4. These samples were homogenized in a Potter-Elvehjem homogenizer in 10 volumes of the 0.32 M sucrose tris buffer solution. 60 Isolation of Subcellular Fractions Portions of the crude homogenate, equivalent to 1 gram of the original tissue, were centrifuged at 850 g for 10 minutes to remove cell debris. The supernate was further centrifuged at 20,000 g to obtain a crude mito- chondrial pellet. The pellet was resuspended in 7 mls of homogenization buffer and 5 mls were layered on a discontinuous sucrose gradient consisting of 1.2 M sucrose and 0.8 M sucrose (Whittaker, 1969). The gradients were centrifuged at 20,000 rpm for 2 hours in a SW22 Rotor with a Beckman Model L2 centrifuge. Synaptosomes (or nerve ending particles) band between 0.8 and 1.2 M Sucrose solutions while the pellet contains partially purified mitochondria. The synaptosomal fraction was carefully removed by pipette and centrifuged at 100,000 g for 90 minutes to pellet the particles. Microsomes were also isolated by centrifuging the 20,000 g supernate fraction at 100,000 g for 90 minutes. Isolation and Determination of the Specific Radioactivity of Glycoproteins Glycoprotein was isolated according to the method of Robinson, Molnar and Winzler (1964). The ether dried glycoprotein material was hydrolyzed in 0.25 N NaOH at 100°C for 20 minutes. Aliquots were counted in a 4 per cent Cabosil scintillation fluid described previously 61 (Chapter II, Materials and Methods) and protein was determined by the method of Lowry, Rosebrough, Farr and Randall (1951). Acid hydrolysis of the glycoprotein pellet from chicks injected with [3H] mannosamine in 0.1N H2804 at 80°C for 30 minutes (Neuberger and Marshall, 1966) released essentially all the radio- activity, indicating that only N-acetyl neuraminic acid was labelled. Isolation and Determination of Radioactivity in Mucopoly- saccharides The glycoprotein fraction, isolated as described above, was treated by modification of the method described by Brunngraber, g£_§1. (1969) for isolation of a muco- polysaccharide fraction. Following digestion of the protein pellet with papain and brief centrifugation of the solution to remove undigested debris, cetylpyridinium chloride was added (2 mg/mg original protein). Samples were allowed to stand for 1 hour at 23°C and then were centrifuged. The supernatant fraction was discarded and the pellet was washed with 5 ml of H20. The precipitate was then resuspended in 2.5 M sodium acetate (10 ml/gm original wet tissue) and 3 volumes of ethanol were added (Katzman, 1971). The precipitate was allowed to collect overnight at -20°C and then, following centrifugation, was dissolved in H20 (6 mls/gm original tissue). Aliquots 62 were taken for radioactive determination (described above) and quantitation of hexosamine (see below). When NaZBSSO4 was used as a precursor, only the crude glycoprotein-containing fraction was measured for 35 35 S. Glyc0protein contains no sulfur; 5 being incor- porated into mucopolysaccharides of this fraction. Isolation of Gangliosides Samples of the crude homogenate equivalent to one gram of original tissue were treated with 5 mls of 10 per cent TCA. The precipitate was resuspended in 6 mls of methanol and 12 mls of chloroform were then added. Extraction of the lipids was carried out at 45°C for 30 minutes in a Buchler Rota-Vap. The tissue was re-extracted chloroform-methanol (1:2, v/v) by the same procedure and the two organic extracts were combined and dried. Lipids were redissolved in 10 m1 of chloroform- methanol (2:1, v/v) and gangliosides were extracted according to the procedure described by Kanfer (1969). The ganglioside fraction was dialyzed overnight against 8 liters of H20 and 1ypholyzed. These samples were redissolved in 4 ml of H O and kept for determinations 2 of hexose, hexosamine, and sialic acid. Quantitation and Specific Radio- activityigetermination of Sialic Acid in Gangliosides Samples were heated for one hour at 80°C in 0.1N H2504 and the sialic acid liberated was measured by 63 the method of Warren (1959). To determine the distribution of tritium in the ganglioside fraction when [3H] mannosa- mine was used as a precursor, the H2504 digest from the two-hour time period (Figure 5) was neutralized with 0.3N ‘ Ba(OH)2 and an aliquot was chromatographed on a Dowex 2 column (5 cm x 0.4 cm diameter, formate form) according to a modification of the method described by Yamashina (1956). The column was washed with 25 mls of H20 to remove desialylated gangliosides and sialic acid was then eluted with 20 mls of 2.4N formic acid. The sample was taken to dryness and the radioactivity was counted. Essentially all the counts applied to the column were eluted in this fraction, indicating that only sialic acid is isotopically labelled. A similar procedure was used when [6-3H] glucosamine was utilized as a precursor (see Results). Quantitation of Bound Hexosamine and Hexose Ganglioside, glycoprotein, or mucopolysaccharide samples were hydrolyzed for 3 hours in 4N HCl. Hexosamine was determined by a modification of the Elson and Morgan method for amino sugars (Boas, 1953). For quantification of glucose or galactose in gangliosides, samples were hydrolyzed in 2N HCl for 3 hours. The HCl was removed by repeated evaporation on a rotary flash evaporator and 64 derivitized for gas chromatography (Sweeley, Wells and Bentley, 1966). a-Methyl mannoside was used as an internal standard. The extent of i§_!iyg_labelling of neutral sugars in gangliosides from intracerebrally injected [3H] glucosa- mine was determined by chromatographing acid hydrolyzates described above on Whatman 3MM paper with a butanol: pyridine: H O solvent (6:4:3, v/v). The region on the 2 chromatogram with an Rf equivalent to standard glucose and galactose was eluted and counted in Bray's scintil- 1ation fluid (109 of 2,5-diphenyloxazole, lOOg-of naphtha- lene and 1 liter of dioxane). No radioactivity above background was detected. Isolation of Free [3H] Mannosamine Aliquots of the crude homogenate equivalent to one gram of brain tissue were deproteinized with an equal volume of 10 per cent TCA and extracted with 4 successive volumes of ether to remove the TCA. The neutralized extract was applied to a Dowex 50X-8 colum (100—200 mesh; 7 cm by 0.9 cm, diameter) in the ammonium form which is known to bind only basic amino acids and amino sugars (Thompson, Morris and Gering, 1959). The column was washed with 30 ml of H20 and [H3] mannosamine was eluted with 25 m1 of 2N NH4OH. The eluent was taken to dryness with a flash evaporator, redissolved in H20 and an aliquot counted in dioxane based scintillation fluid. 65 Results Incorporation of [3H] Glucosamine Into Gangliosides Gangliosides from brain tissue homogenates of chicks injected intracranially with [6—3H] glucosamine were extracted and their specific radioactivities deter- mined (Figure 6). Initially, tritium was incorporated faster into the gangliosides of the galactose-fed animals but by 60 minutes the total amount utilized was slightly less than that observed in control chicks. The percentage of the total radioactivity present in the form of sialic acid was approximately 30 per cent for both experimental and control animals at 20 and 60 minutes. Since no radio- activity could be detected in glucose or galactose from gangliosides, it was concluded that approximately 70 per cent of the tritium was present as galactosamine. The levels of glucose, galactose, galactosamine, and sialic acid were measured and found to be essentially unaltered in the galactose-fed chicks (Table 3). Biosynthesis of Mucopoly- saccharides The utilization of [3H] glucosamine for muc0poly- saccharide synthesis differed slightly from that observed for gangliosides (Figure 7). Tritium incorporation was approximately 40 per cent faster in the galactose intoxi- cated chicks than in controls for the first 30 minutes. 66 However, there was a leveling off of tritium incorporation in the control animals by 60 minutes while the rate remained unchanged in the experimental animals. Levels of hexosamine in the mucopolysaccharide fraction were not found to be statistically different between the two dietary groups (Table 3). In contrast, the incorporation rate of NaBSSO4 into Chondroitin sulfate, a subclass of mucopoly- saccharides, in galactose—fed chicks was essentially identical to that of controls (Figure 8). It is gen- erally accepted that sulfation of hexosamine residues of acid polysaccharides occurs as the polysaccharide chain grows (White, Handler and Smith, 1968). This suggests that augmented flux rate of [3H] glucosamine into chon- droitin sulfate is not due to an actual increase in the rate of biosynthesis although sulfation could possibly lag behind an increased glycosylation rate. A third study was attempted using [14C] glucuronic acid as a precursor but no radioactivity was detected in a trichloroacetic acid precipitate two hours after intra- cerebral injection. Incorporation of [6-3H] Mannosamine Into Glyc0protein and Gangliosides As seen in Figure 9, [3H] mannosamine incorporation into glycoprotein was biphasic and markedly faster in the galactose-fed chicks. Over the first 60 minutes the rate 67 was three times that of the control animals. Likewise, the ganglioside fraction was labelled faster in the eXperi- mental chicks, although the difference in the initial rate was not as great as in the glyc0protein fraction (Figure 10). Figure 6 demonstrates the labelling of glycoprotein in the subcellular fractions. The micro- somal fraction (Figure 11, B) had the highest specific radioactivity in both control and experimental groups. In both the mitochondrial and microsomal fractions from galactose-fed chicks, [3H] mannosamine incorporation was twice that found in controls over the first 60 minutes while in the synaptosomal fraction the rate of labelling was somewhat less. In Table 4 are tabulated the levels of radio- activity in the TCA soluble and free mannosamine fractions. As can be seen, mannosamine appears to be metabolized faster in the galactose-fed chicks. However, the total radioactivity decreases faster in the control brain (Table 4, TCA soluble fraction at 120 minutes) suggesting that some of the radioactivity in the brain of control animals may be equilibrating faster with the blood at later times. Discussion Uridine diphospho N-acetylglucosamine (UDPAG) is the common intermediate for N-acetylglucosamine transfer into glycoprotein and mucopolysaccharides. 68 UDPAG 4-epimerase (E.C.5.l.3. 2d) converts UDPAG to UDP N-acetylgalactosamine, the immediate donor in ganglioside and glycoprotein biosynthesis.. A faster synthesis rate of any one or two of these glycomacro- molecules in the galactose-fed chick would in turn affect a faster turnover rate of the common metabolic pools in the pathway from glucosamine to UDPNG or UDP N-acetylgalactosamine. Introduction of radioactively labelled glucosamine under such conditions would result in a more rapid equilibrium of the tracer with the uridine amino sugar intermediates and give the appearance of a greater initial rate of incorporation of radioactivity into all fractions when compared with control animals under normal conditions. This phenomena may explain the [3H] glucosamine incorporation into the mucopolysaccharide fraction (Figure 7) which, like that shown for gangliosides (Figure 6) and glycoprotein (Chapter II, Figure 4), is observed to occur at a faster rate in the galactose-fed chicks. Normal 35 SO4 utilization in the animals (Figure 8) suggests that chondroitin sulfate synthesis is not altered and the enhanced flux of [3H] glucosamine into the whole acidic polysaccharide fraction could well be secondary to increased demands on UDPAG for glycolipid and/or glycoprotein biosynthesis. 69 The levels of sialic acid measured in glycoprotein (Table 3) agree fairly closely with those found in rat brains (1.2 umoles/gm tissue) by Quarles and Brady (1970). Garrigan and Chargaff (1963) found sialic acid levels in gangliosides from 2-day-old chicks to be approximately 2.0 umoles/gm tissue which is somewhat higher than those detected in these studies (Table 3). When expressed on a molar basis, the ratios of glucose:galactose:galactosa- mine:sialic acid in gangliosides are l:l.9:l.3:2.3 respectively and are in close agreement with the theoreti- cal ratios of 1:2:1 for glucose:galactose:ga1actosamine. The pathway for conversion of mannosamine to N-acetylneuraminic acid has not been elucidated in the brain. It is presumed that mannosamine is phosphorylated and acetylated to N-acetylmannosamine 6-phosphate (a sequence parallel with glucosamine metabolism) where it enters the established pathway for conversion of UDPAG to CMP-Nacetylneuraminic acid, the latter of which is the sialyl donor to gangliosides and glyco- protein. Raisys and Winzler (1970) demonstrated phos- phorylation of the 6 position of mannosamine in Sarcoma 180 cells. However, no further metabolic products were detectable. In the same study, neither rat liver nor kidney exhibited any metabolic activity toward [14C] mannosamine. Most of the radioactivity was excreted in the urine as [14C] mannosamine within two hours 70 following intraperitoneal injection. Hence, mannosamine conversion to sialic acid in the brain appears to be unique to the tissues studied to date. A specific kinase and transacetylase could be involved in its metabolism, although the normally low levels or absence of free mannosamine in the brain would suggest that a nonspecific kinase and acetyltransferase could play the needed cataly- tic roles. The enhanced incorporation rate of [3H] mannosa- mine into glycoprotein and glycolipid of the neurointoxi- cated chicks in consistent with the earlier observations of increased [3H] glucosamine utilization for ganglioside and glyc0protein synthesis. Since protein and lipid bound amino sugars do not appear to be altered in whole brain, a faster turnover rate of one or both of these fractions remains a possibility. The more rapid metabolism of free [3H] mannosamine could be interpreted as a response to the greater require- ments for sialic acid incorporation to glycomacromolecules in the galactose treated animals. However, if mannosamine is phosPhorylated by a hexokinase not under regulatory control by mannosamine 6-phosphate or other intermediates in the sialic acid biosynthetic pathway, a more trivial eXplanation could be advanced for the observation. Brain glucose levels in the galactose-fed chick are markedly lower than in controls (Kozak and Wells, 1969). 71 [3H] Mannosamine competition with different pool sizes of glucose for the same enzyme could be reflected in a faster phosphorylation rate for the amino sugar in the smaller glucose pool, free mannosamine metabolism being then a function of intracellular glucose concentrations. Several lines of evidence argue against this possibility. Raisys and Winzler (1970) found that mannosamine 6-phosphate formation in Sarcoma cells was faster in the presence of glucose than in its absence. In their subsequent studies with rat liver and kidney, hexokinase was unable to phosphorylate mannosamine. The turnover rate of [14C] glucose has been measured in chick brain and appears to be much slower in the galactose-fed animals than controls suggesting that hexokinase turn- over number in vizg is actually slower in the brains of the galactose neurotoxic chicks (Granett, Kozak, McIntyre and Wells, 1972). Other radioactive precursors for glycoprotein and gauglioside biosynthesis such as glucose and galactose suffer from the disadvantages of alternate pathways by which they can be metabolized and a large disproportion- ation in the intracellular levels between experimental and control chicks. Although the mannosamine tracer studies support the concept of a faster turnover of glycoprotein and gangliosides, a lack of knowledge of its route of metabolism and the levels and Specific 72 activities of the intermediates in its metabolism pre- cludes this as conclusive evidence. The possible role of lysosomal enzymes in contributing to abnormal glyco- protein and ganglioside metabolism will be discussed in the following chapter. REFERENCES Barondes S. H. in The Neurosciences Second Study_Program (Edited by F. 0. Schmitt), p. 747. The Rocke- feller University Press, New York (1970). Boas N. F., J. Biol. Chem. 204, 553 (1953). Brunngraber E. G., Brown B. D. and Aguilar V., J. of Neurochem. 16, 1059 (1969). Crandall M. A. and Brock T. D., Science 161, 473 (1968). Garrigan O. W. and Chargaff E., Biochem. Biophys. Acta. 10, 452 (1963). Gesner B. M. and Ginsberg V., Proc. Nat. Acad. Sci. 52, 750 (1964). Ghosh S. and Roseman S., Proc. Nat. Acad. Sci. 41, 955 (1961). Granett S., Kozak L. P., McIntyre J. and Wells W. W., Submitted for publication (1972). Kanfer J. N. in Methods in Enz molo (Edited by J. M. Lowenstein), Vol. XIV, p. 66%. Academic Press, New York (1969). Katzman R. L., J. of Neurochem. 18, 1187 (1971). Kozak L. D. and Wells W. W., Arch. Biochem. Biophys. 35, 371 (1969). Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J., J. Biol. Chem. 193, 265 (1951). Neuberger A. and Marshall R. D. in Glycoproteigs (Edited by A. Gottschalk )1 p. 216. Elsevier Publishing Co., New York (1966). 73 74 Quarles R. H. and Brady R. 0., J. of Neurochem. 11, 801 (1970) . Raisys V. A. and Winzler R. J., J. Biol. Chem. 245, 3203 (1970). Rutter W. J., Krichevsky P., Scott H. M. and Hansen R. G. Poultry Sci. 3;, 706 (1953). Sweeley C. C., Wells W. W. and Bentley R. in Methods in Enzymology (Edited by E. E. Nuefeld and V. Ginsburg), Vol. VIII, p. 95. Academic Press, New York (1966). Thompson J. F., Morris C. J. and Gering R. F., Analyt. Chem. 31, 1028 (1959). Warren L., J. Biol. Chem. 234, 1971 (1959). White A., Handler P. and Smith E. L. Principles of Bio- chemistry, 4th Edition, p. 883. McGraw-Hill Book Company, New York (1968). Whittaker V. P. in Handbook of Neurochemistry (Edited by A. Lajtha), Vol. II, p. 327. Plenum Press, New York (1969). Yamashina I., Acta. Chem. Scand. 10, 1666 (1956). 75 TABLE 3.--Levels of Neutral and Amino Sugars in Ganglio- sides, Glycoprotein and Mucopolysaccharides. Levels nmoles/brain i s.d. Control Galactose Gangliosides Sialic Acid 1380 i 82 1440 i 76 Galactosamine 840 i 53 907 i 50 Galactose 1130 i 609 1240 i 100 Glucose 640 t 35 600 i 40 Glycoprotein Sialic Acid 1500 i 100 1400 i 160 Hexosamine 4500 i 400 4700 t 600 Mucopolysaccharide H- H- Hexosamine 154 20 139 12 76 TABLE 4.--Change in Radioactivity in Free Mannosamine and the TCA Soluble Fraction with Time Following Intracerebral Injection of l uCi of [6-3H]-D-Mannosamine. Time Mannosamine (in dpm) TCA Soluble (in dpm) Control Galactose Control Galactose 30 825,000 550,000 1,071,000 1,208,000 60 603,600 393,000 814,000 1,286,000 120 264,400 132,000 480,000 996,000 Above values are subject to approximately 10 per cent range of variability. 77 Figure 6. Incorporation of [3H] galactosamine into gangliosides. Gangliosides, purified from homogenates of brain tissue as described in methods, were re— dissolved in H2( and duplicate aliquots counted in Bray's scintillation fluid. Separate aliquots were hydrolyzed and galactosamine content determined. Range of error < 10 per cent. Control, O————O; Galactose, O----O. 78 658320260 0.2:: \zao n..o_ 60 40 ‘20 Time (min) Figure 7. 79 Incorporation of [3H] hexosamine into muco- polysaccharides. Vertical bars indicate the range of error in determining the specific radioactivity from two separate extractions of the acid poly- saccharides. Control 0————O; Galactose O----O. 80 _ 0 0 3 056338: a: \ sin 500 "' Time (min) Figure 8. 81 35504 incorporation into chondroitin sulfate. Radioactivity was determined in triplicate aliquots from the crude homogenator as described in Methods. Standard deviation 1'10 per cent. Control O————O; Galactose O----O. 82 IZO 522.. 2.. \8m 3 2.8 N.o. Time (min) I 'H'l'” -' Figure 9. 83 [BK] Mannosamine incorporation into glyco- protein as sialic acid. Triplicate aliquots from the crude homogenates of each time period indicated were assayed as described in Methods. Standard deviation approximately 10 per cent. A second [3H] mannosamine incorporation study gave essen- tially identical results. Control O————C; Galactose O----O. 84 500 " _ O O 3 5293 as \ 5E0 60 9O |20 Time (min) 30 :4 Figure 10. 85 [3H] Mannosamine incorporation into ganglio- sides as sialic acid. Hydrolyzates of the ganglioside fraction were neutralized and duplicate aliquots taken for determination of radioactivity and sialic acid. Range of error approximately 15 per cent. Control 0_———O; Galactose O—---O. 86 28 2:5 22.5 \ 2%. mo. Time (min) 87 .Ammaouwov mmEomoummchm .Ammamsmauuv mweomou0wz um Inomm mmouomamm .moaouflo ammo «Houucoo .mmaouflo omuoHou .ucoo umm ma v nouns mo omcmm .mumowHQSU ca owumuwucmov memEMm Had .mcofiuomum umHoHHmonom mo samuoumoomam ouca wcHEMmoscmz ”mm“ mo cofiumuomuoocH .HH magmas 600 DPM / mg protein 200 Mitochondrial 88 B. Microsomal and Synaptosomol , Time (min) CHAPTER IV ENHANCED FRAGILITY OF NEURAL LYSOSOMES FROM CHICKS SUFFERING FROM GALACTOSE TOXICITY Abstract The stability of neural lysosomes to osmotic and temperature shock and the free (non-sedimentable) activi- ties of selected lysosomal hydrolases from chicks suffer- ing from galactose neurotoxicity were investigated. The neural lysosomes from chicks fed galactose demonstrated enhanced fragility to both elevated temperature and hypo- osmotic media in comparison to the behavior of neural lysosomes isolated from control animals. The increased lability to osmotic shock could be duplicated by pre- incubation of normal lysosomes in solutions of galactose or galactitol. Further, the increased fragility induced i2_giyg by galactose feeding could be reversed by removing the chicks from the diet for 8 hours, and such removal was accompanied in the brain by large reductions in levels of galactose and galactitol. The free activities of both B-galactosidase (EC.3.2.1.23) and B-N—acetyl hexosaminidase (ED.3.2.1.30) were elevated above those of controls, and the percent increases were proportional 89 90 to the combined brain levels of galactose and galactitol. Our data suggest that increased fragility of lysosomes is a function of the accumulation of galactose and galactitol in the brains of chicks fed toxic amounts of galactose. Alteration of lysosomal integrity represents an attractive role for galactitol, as well as galactose, in the causation of galactose neurotoxicity in chicks. Introduction Recent studies in our laboratory (Knull, Blosser, and Wells, 1971: Blosser and Wells, 1972) have demon- strated a faster rate of incorporation of [3H] glucosamine into glycoprotein in galactose-fed chicks, while protein bound glucosamine levels remained unchanged, an obser- vation suggestive of a faster rate of turnover of protein- bound glucosamine. Such a phenomenon could well involve dysfunction of lysosomal degradative processes. It is characteristic of galactose toxicity in chicks fed a diet containing 40 per cent D-galactose that there is accumulation of significant levels of galactose and galactitol (Kozak and Wells, 1971). Aldohexoses and hexitols have been shown to permeate lysosomes from rat liver (Lloyd, 1969) and from Tetrahymena pyriformis (Lee, 1970). This effect is associated with a decrease in the latent (detergent-activated) activity and a cor- responding increase in the free activity of various acid 91 hydrolases from lysosomal preparations. When cell cultures are incubated in the presence of aldohexoses, hexitols or disaccharides, the acid-phosphatase-containing particles become extensively vacuolated (Dingle, Fell, and Glauert, 1969; Nyberg and Dingle, 1970). Our pre- sent study was undertaken to assess the effects of galactose and galactitol on the stability of neural lysosomes and on the free activities of selected lyso- somal acid hydrolases. Materials and Methods Animals and Materials Day-old Leghorn cockerels were purchased from either Cobbs, Inc. (Goshen, Ind.) or Klagers (Manchester, Mich.) and kept in a brooder at 32°C. Animals were placed on synthetic diets described by Rutter, Krichevsky, Scott, and Hansen (1953) containing 40 per cent (w/w) D-galactose in the place of a corresponding quantity of cerelose. The substrates used in the assays of the enzymes examined were: p-nitrophenyl B-D-galactopyranoside for B—galacto- sidase (B-D-galactoside galactohydrolase; EC 3.2.1.23), p-nitrophenyl-2-acetylamino-2-deoxy-B—D-glucopyranoside for N-acetyl hexosaminidase (B-2-acetylamino-2-deoxy-D- glucoside acetylaminodeoxy glucohydrolase, EC 3.2.1.30), and p-nitrophenyl phosphate for acid phosphatase (ortho- phosphoric monoester phosphohydrolase, EC 3.1.3.2), all 92 purchased from Sigma Chemical Co. (St. Louis, Mo.). Other chemicals were of analytical grade. Preparation of Lysosomal Fraction Brains were removed from decapitated chicks and placed in 10 m1 of ice-cold homogenizing solution (250 mM sucrose with 1 mM EDTA). All of the following prepara- tive operations were carried out at 0-4°C. The solution was discarded and the brains were minced with a razor blade. The tissue was then placed in a Potter-Elvehjem homogenizer in 8 vol of homogenizing solution and dis- rupted by three up-and-down strokes with a loose-fitting, motor-driven pestle at slow speed. The resulting homogen- ate was centrifuged at 800 g for 10 minutes to remove cell debris. To obtain a crude lysosomal pellet, the 800 g supernatant fluid was then centrifuged at 20,000 g for 15 minutes. Enzyme assays were carried out on the post lysosomal supernatant fraction or the crude lysosomal fraction, which was gently resuspended by hand in 4 vol of the appropriate carbohydrate solutions (see Legends to Figures) with a Potter-Elvehjem homogenizer as already described. Enzymatic Assays A11 enzymatic activities were assayed according to a modification of the method described by Frohwein and Gatt (1969) for B-N-acetyl hexosaminidase. The 93 reaction mixture contained in final concentrations: 0.8 mM for the appropriate substrate, 50 mM sodium phosphate-citrate buffer (pH 5.0, or for the B-N-acetyl hexosaminidase assay, pH 3.8); homogenate; and water to 0.5 ml. Homogenate was added at several concentrations to the assays to substantiate linearity of the reaction rate with respect to enzyme concentration. For esti- mation of total sedimentable enzyme activity, 20 ul of 5 per cent (w/v) Triton X-100 were added to a reaction mixture containing portions of the resuspended crude lysosomal pellets. All assay mixtures for enzymes were incubated in a 37°C water bath for 1 hour except for acid phosphatase, in which case the incubation was for 20 minutes. The reactions were stopped by addition of 1 m1 of 2.7 per cent (w/v) TCA, and the mixtures were centrifuged for clarification and neutralized with 0.2 m1 of 1 N NaOH. Sodium borate (1.3 m1 of a 0.125 M solution) was added, and p-nitrOphenol was measured at 410 nm 3 1 = 15.5 X 10 M- cm-l) with a Gilford 300 spectro- (€410 photometer. Protein was estimated according to the method of Lowry, Rosebrough, Farr, and Randall (1951). Free or released enzyme activity refers to that which did not sediment at 20,000 g for 15 minutes. Total activity refers to that detected in the 800 g supernate in the presence of 0.2 per cent (w/v) Triton X-100, whereas total sedimentable activity is defined as activity 94 measured in the 20,000 g resuspended pellet in the presence of 0.2 per cent (w/v) Triton X 100. Soluble activity refers to that remaining in the 100,000 g supernatant. Quantification of Galactose and Galactitol Portions of the crude homogenates were depro- teinized by the Somogyi technique and assayed for galac- tose and galactitol by gas-liquid chromatography (Sweeley, Wells and Bentley, 1966) or for galactose by Galactostat (Worthington Biochemical Corp., Freehold, N.J.). Essen- tially identical amounts of galactose were detected by either method. Results Stability of Lysosomes to Osmotic and Temperature—Shock To investigate possible alterations in the structural integrity of lysosomes during galactose neurotoxicity, crude lysosomal fractions were prepared from brains of chicks fed experimental or control diets for 48 or 64 hours and the fractions were subjected to hypotonic sucrose solutions. The enhanced release of B-N-acetyl hexosaminidase, acid phosphatase, and B-galac- tosidase from the lysosomes of galactose—intoxicated chicks (Figure 12) reflected an increased susceptibility to osmotic shock. That the increased release of activity 95 was not attributable to more total enzyme could be shown by demonstration of equal activities of all three enzymes in both dietary groups in lysosomal fractions resuspended in isotonic sucrose and assayed in the presence of 0.2 per cent (w/v) Triton X-100. Further evidence for increased instability of neural lysosomes from galactose-fed chicks was obtained by the demonstration of enhanced release of B-N-acetyl hexosaminidase from lysosomes preincubated at various temperatures (Figure 13A). Increased temperatures affected acid phosphatase to a lesser extent (Figure 133); however, more enzyme was released from the lysosomes of the galactose-fed animals. B-Galactosidase did not appear to be released to any appreciable extent, even with tem- peratures up to 40°C. Stability of Lysosomes Preincubated in Galactose or Galactitol to Osmotic Shock To ascertain whether galactose or galactitol alone could cause the increased lability, lysosomes from con- trol chicks were preincubated in galactose, galactitol, or sucrose solutions, supplemented with sucrose for a total concentration of 250 mM, and then subjected to osmotic shock. As illustrated in Figure 14, preincu- bation with galactitol and, to a slightly lesser extent, with galactose (50 mM concentrations) had an appreciable effect on the release of both B-N-acetyl hexosaminidase 96 and B-galactosidase. As control experiments, galactose and galactitol at similar concentrations were added to enzymes released by detergent or H20 and were shown to have no stimulatory or inhibitory effect on the activities of either of these enzymes. Similarly, when compared with isotonic KCl, 250 mM sucrose did not affect any of the enzyme activities. Although B-N-acetyl hexosaminidase was the most active of the enzymes studied, water extracted only 15 to 25 per cent of the total activity (Figure 14A). When pellets of water-shocked lysosomes were resuspended and assayed for B-N-acetyl hexosaminidase in the presence of Triton X-100, 75 and 85 per cent of the original total activity was detected as still bound to the particles from the galactose and control groups, respectively. On the other hand, over 85 per cent of B-galactosidase and 50 to 60 per cent of the acid phosPhatase activity was released by osmotic shock in H20. In contrast to the increased fragility to osmotic shock, control lyso- somes preincubated with 50 to 100 mM galactose or galacti- tol for 45 minutes at temperatures from 0 to 38°C did not exhibit increased lability (as monitored by release of acid phosphatase or B-N-acetyl hexosaminidase) in com- parison to those samples incubated similarly in sucrose. To determine whether the increased lability of lysosomes was reversible, chicks were fed the diet con- taining galactose for 44 hours and then placed on the 97 control diet for 8 hours. Stability of lysosomes from “recovered" animals closely approximates those of controls (Figure 15), except at extreme hypotonicity where the extent of fragility was intermediate between that of lysosomes from control and those from galactose-fed chicks. Levels of galactose and galactitol in the galactose-fed animals were 8.8 and 6.4 umoles/g wet weight of tissue, respectively, whereas in the "recovered" animals, galactose could not be detected and levels of galactitol had dropped to 3.5 umoles/g of tissue. Activities of Free Acid Hydrolase as a Function of In Vivo Levels of Galactose and Galactitol Chicks fed galactose or control diets were sacri- ficed 18 to 64 hours later and the post-lysosomal super- natant fractions were prepared from the brains and assayed for B-galactosidase and B-N-acetyl hexosaminidase. The results of 7 experiments are summarized in Table 5. The activities of both enzymes from the brains of galactose— fed chicks were elevated above those measured in prepar- ations from controls. The variation in the levels measured did not correlate directly with increasing time periods of dietary feeding or increasing levels of galactose or galactitol alone. However, a good proportional relation- ship appeared to exist between the enzyme activities and the sum of the levels of galactose and galactitol. (The calculated correlation coefficients (Steel and Torrie, ind 98 1960) were 0.82 for B-galactosidase and 0.85 for B-N- acetyl hexosaminidase which are significant at the 5 and l per cent levels, respectively. The amount of free activity for B-galactosidase and B-N-acetyl hexosamini- dase was low and typically 5 to 9 per cent of the total activity. In similar studies, in which galactitol levels were not measured, there was little or no increase in the free activities of acid phosphatase, whereas B-galactosi- dase and B-N-acetyl hexosaminidase exhibited increases in free activity similar to those presented in Table 5. To determine the degree of solubility of the released enzyme activity measured in the post-lysosomal supernatant fractions, portions of these preparations from the 43 and 64 hour periods (Table 5) were centrifuged at 100,000 g for 60 minutes. Approximately 65 per cent of the B-galactosidase remained soluble (Table 6). How- ever, the difference in the enzyme levels between galactose- fed chicks and their corresponding controls compared closely with those differences seen in the reSpective 20,000 g supernatant fractions. This would indicate that the increases in the levels of B-galactosidase observed in the 20,000 g preparations from the galactose-intoxicated animals are due to enzyme released from the lysosomes and not the result of increased gg_ngyg_synthesis of the enzyme in the microsomes. Previous studies-have indi- cated no alteration in general protein synthetic rates 99 in the galactose-intoxicated chicks (Blosser and Wells, 1972). Only 5 to 8 per cent of the B-N-acetyl hexosamini- dase found in the 20,000 g supernatnat fractions was recovered in the 100,000 g supernate although the levels from the experimental animals were markedly higher than those measured from controls (see Discussion and Table 6). Plasma levels of N-acetyl.hexosaminidase, B-galac- tosidase, and acid phosphatase in galactive and control- fed chicks were measured to ascertain whether acid hydrolases might be released by tissue as a result of cell damage. As seen in Table 7, concentrations of free acid hydrolases are essentially identical from the con- trol and experimental chicks. Discussion As indicated by the enhanced release of lysosomal enzymes by osmotic and temperature shock, lysosomes from the brains of galactose-fed chicks are more fragile, presumably as a result of the uptake and concentration of galactose and galactitol by the lysosomes. This latter conclusion is based on (1) the demonstration of enhanced lability of normal lysosomes to osmotic shock after preincubation in galactose or galactitol, (2) a correlation between the sum of in yizg levels of galac- tose and galactitol and the increase in free activities of B-galactosidase and B-N-acetyl hexosaminidase, and (3) the loss of increased fragility accompanying 100 reduction of levels of galactose and galactitol in the brain by 8 hours after the animals were removed from the experimental diet. The enzymes studied exhibit differential responses to osmotic or temperature shock (Figures 12 and 13). Such phenomena have been observed previously with these or other acid hydrolases in lysosomes from rat liver (Baccino, Rita and Zuretti, 1971) or rat brain (Sellinger and Nordman, 1969; Sellinger and Hiatt, 1968; Bowen and Radin, 1969). These latter investigations have suggested differential structural affinities of the acid hydrolases for the lysosomal matrix and, hence, a variation in the ease with which disruptive agents may effect solubili- zation of different hydrolases. Following disruption of brain tissue in hypotonic sucrose and differential centrifugation, Sellinger and Nordman (1969) concluded that much of the B-N-acetyl hexosaminidase was still bound to small membrane fragments of disrupted lysosomes, and in adsorbability experiments, Bacinno §£_gl. (1971) demonstrated that previously solubilized B-N-acetyl hexo- saminidase was almost completely readsorbed to particu- late material from rat liver homogenates. Such a high degree of membrane adherence by the soluble enzyme could explain the loss of most of the activity of the B-N- acetyl hexosaminidase in the 20,000 g supernatant fraction in our studies when this fraction was further centrifuged at 100,000 g for 1 hour. 101 One inconsistency between the behavior of lyso- somes from galactose-fed chicks and those from control animals preincubated with galactose in galactitol is the apparent absence of an augmented heat lability in the latter case. This difference may reflect an insufficient concentration of galactose or galactitol in the supple- mented control lysosomes for enhanced reaponse to a rather gentle disruption procedure such as elevated temperature. There also may be a difference in the mode by which lysosomes become fragile i§_!i!9 and in yitgg. In yiyg, increased lability could result, in part, from the vacuolation produced by monosaccharides in the lysosomes (Dingle g£_§1., 1969), whereas in 25552, the much shorter incubation periods with monosaccharides may cause simple swelling of the lysosomes by imbibition of water in response to the inward diffusion of mono- saccharides (see Lloyd, 1969). Hence, distortion of the lysosomal matrix in different manners could affect the responsiveness to different types of stress. Perhaps the relative lengths of exposure of lysosomes to galactose and galactitol compounded with radically different environments in_git£2 and in_yiyg_may also explain the need for levels of monosaccharides in_yi££9 several times those attained in giyg_to elicit a similar response to osmotic shock. 102 From present experiments, it is difficult to ascertain whether the increases in free enzyme activities in brains of galactose-fed chicks (Table 5) represents leakage of the enzymes from the lysosome ig_yigg or release that occurs during homogenization of the tissue as a result of increased fragility. Even though most of the B-N-acetyl hexosaminidase was observed to be membrane bound, it could be argued either that it comes from the ruptured lysosomes, supporting the release-during- homogenization interpretation, or that it represents enzyme released in_viyg_which had adhered to, for example, microsomal membrane. The lack of increase of free levels of acid phosphatase over control free values might suggest a localization in or affinity to the lysosomal matrix such that acid phosphatase cannot be released readily in gi!g_or, on the other hand, the lack of increase might suggest that the careful pro- cedures used for disruption of the tissue were not sufficient stress to enhance the release of the enzyme from more fragile lysosomes. An abnormal release of acid hydrolases from lysosomes has been implicated in the pathology of a variety of disorders, including rheumatoid arthritis (reviewed by Lack, 1969), muscular dystrOphy (reviewed by Weinstock and Iodice, 1969), vitamin E deficiency (reviewed by Roels, 1969), and poisoning by such agents 103 as vitamin A, bacterial toxins, or UV irradiation (reviewed by Slater, 1969). Whether increased lysosomal fragility or possible leakage of hydrolases into the cytoplasm would disrupt the cellular degradative pro- cesses in galactose neurotoxicity remains to be eluci- dated. However, independent findings lend support to such a possibility. Neurohistological studies on chicks during galactose toxicity demonstrated degeneration of neurons in the basal ganglia, medulla, and occipital lobes (Rigdon, Couch, Creger and Ferguson, 1963). Levels of hexosamine in mucopolysaccharides and, in gray matter, in glycoprotein were found to be significantly lower from an individual suffering from human hereditary galac- tosemia than those found in normal adults (Haberland, Perou, Brunngraber, and Hof, 1971). Another neurologic disorder, human neurolathyrism, is caused by ingestion of B-N-oxalyl-L-a,8 diamino propionic acid from the seeds of Lathyrus sativus. Injection of this amino acid into animals is thought to increase catabolism of protein and nucleic acids and has also been shown to increase the free levels of acid phOSphatase, ribonuclease and cathep- sin, all 1ysosomal enzymes (Lakshmanan, Cheema and Pad- manaban, 1971). Disruption of lysosomal integrity and increases in the free levels of acid hydrolases is supportive of the hypothesis of a faster turnover of carbohydrate 104 constituents of glycolipid and glycoprotein. That free levels of acid phosphatase did not increase over control values while the levels of the other two enzymes studied did, suggests that not all lysosomal enzymes would exhibit increased free activities in the brains of galactose-fed chicks. Several additional acid hydrolases have been measured, using p-nitrophenol substrate derivi- tives, and were found to be absent or present in low levels in lysosomal fractions from chick brain. Activi- ties of a-L fucosidase, B-glucuronidase (E.C.3.2.l.31) and 8-D mannosidase (E.C.3.2.l.24) were quite low in comparison to B-galactosidase while arylsulfutase (E.C.3.l.6.1) and B-glucosidase (E.C.3.2.l.21) could not be detected in the lysosomal fraction in the presence of Triton X-100. With the exception of B-glucuronidase, none of these enzymatic activities were found in the 20,000 g supernatant fraction. This would suggest that increased catabolism of only specific types of glycosidic bands or cellular macromolecules might be expected to occur during galactose neurotoxicity. REFERENCES Baccino F. M., Rita G. A., and Zuretti M. F. Biochem. J. 122, 363 (1971). Blosser J. and Wells W. W., J. Neurochem. 13, 69 (1972). Bowen D. and Radin N. S., Biochim. Biophys. Acta (Amst.) 152, 599 (1968). Dingle J. T., Fell H. B. and Glauert, A., J. Cell Sci. '4, 139 (1969). Frohwein K. Z. and Gatt S. in Methods in Enzymology (Edited by J. M.~LoWenstein), Vol. XIV, p. 161. 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North-Holland Publishing Co., Amsterdam-London (1969). 107 .ucmo mom m w muHHHnmwum> mo omcmn m saws oumowamwuu ca oocwauouoo muoz coauomnm oumnmmom some so mammmd .cwmun no me you no manna» nmoum mo Edam mom oommmumxo Hmnuos3 oo>nomno mums moan Ifl>fiuoo oauaoomm ca mmocoummmao .>Ho>fluoomnou .m.a n h.ma one pm A com ouo3 ommoamou looaomlm can omoofioafimmoxon HauoUMIZIm Mom A.o.m Hy monam> Houucoo coo: .oohmmmm one .Amoonuoz oomv ooummoum mcowuomum ucmumcuomom u ooo.om one .ooaoom ouo3 msfloun “Hosp «nouoowocfl moEwu on» no conflmauoom ouo3 mooum mumuoflo some Eoum mxowno Hsom m.H¢ 5mm o.ma m.m m.> . we 0.5m va «.ma m.m h.m mv «.mm omm o.m m.~ m.m mm m.m~ mum o.m o.v o.v mv m.m~ mmm ¢.h ¢.m o.v Nv m.m~ «mm «.5 v.H o.m ma m.m~ mum m.m m.H m.v ma momma» me ooa\£\aoco£mouufislo oommwu m\HoE: n Houauomamw smooam moccasaeomoxom . . . . + HoufluomHoO omouomaow uofio so mafia nouomaeoum Haumo4uznm mmouomaeo .uowa mmouomaou nmflm m ooh mxownu mo msflmum cw mmooacafimmoxmn Hmumodlzlm.mmm ommpflmou nomamonm «0 m0nua>fiuoa 000m use Houfiuomamo can mmouomaeo mo mH0>0q o>fl> :Huu.m mamas 108 TABLE 6.--Soluble Activities of B-Galactosidase and B-N- (Acetyl hexosaminidase in Brains of Chicks Fed Control or Galactose-Containing Diets. 4 fr . Galactose . ngzton + hegogwggfiiggse B-Galactosidase Galactitol h nmol/g nmoles p-nitrophenol . tissue released/h/lOO mg tissue Control Galactose Control Galactose 43 12.2 12 27 9 18 64 13.0 12 37 11 32 Triplicate assays were made on each separate fraction with a range of variability g 5 per cent. 109 TABLE 7.--Levels of Selected Acid Hydrolases in Plasma. Control Galactose (nmoles p-nitophenol released/ r m1 plasma) ‘ B-N-acetylhexosaminidase 876 i 200 (2) 866 i 107 (5) ‘ B—Galactosidase 135 i 5 (2) 131 i 18 (5) Acid phosphatase 105 i 8 (3) 98 i 10 (3) Chicks were anesthetized with dithylether and exsanguinated with a heperinized seringe. Blood was pooled from groups of 4 chicks and centrifuged to remove red blood cells. The plasma was assayed for the enzymes indicated as outlined in methods. Values are expressed i s.d. The numbers in parentheses represent the number of pools assayed. [.4 110 .ucoo mom m mHouoEonummo mo3 muHHHQMHum> mo omcmm .mmmmmo oUMOHHQso mo omouo>m on» mucomoummu ucHom comm .ommuunmmonm oHom mom coulomOHUMHmm Giulio Houucoo Ollllo |.O .ommoHcHEomoxonHauooeizlm Mom coulomouomHmm onlluo Houusoo OIIIIO .m .omoonouomemim How oomnomouoonm Giulio Houucoo OIIIIC .< .mGOHuoonm usouosuomsm map so some mums masons season .mmuscHE OH How d.ooo.o~ um oomstuucooou can .Ooo um :oHusHom omOHUSm oumHumoummm on» NO HE v cH poocomnomou .osmmHu nmoum «o m hm.o aHouoEonummm on mow uncommouuoo mcoHuuom Hosqo ousH oooH>Ho .moonuoz cH oonHuomoo on wound loud ouo3 museum mumuoHo 03» on» no some Eoum mHOHno m Eoum mmEOmomMH .mchun HUHno Eoum mmEomommH mo muHHHnmun co xoonm OHuoEmo mo pomwmm «mi. .NH muomHm 111 5.0 : Tossing 00.0 2.0 . 0N0 .0.0 00.0 on. con _ _ _ _ _ H _.( _ 0mm imam /moq/}oueqdou|u-d mom 1 00¢ I. 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O l O. 05 L 0.10 [Sucrose] M l O.l5 800 L 1 l l o o o o «o o 8 fi' ones): bwom /.moq / |oueqdomu-d se|0wu SUMMARY Rates of synthesis of protein and carbohydrate containing macromolecules do not appear to be decreased in response to the moderate reductions in ATP and phos- phocreatine levels and the energy charge in the brains of galactose-fed chicks. This lack of effect is in con- trast to suggestions by other workers on the influence of the levels of ATP and energy charge on protein synthe- sis rate (see Chapter II, Introduction). It appears that under conditions of these studies, biosynthesis of these macromolecules have high priority with respect to utili- zation of available energy reserves. These findings do not exclude the possibility of an individual protein having an altered synthesis rate. However, such a change in the normal synthesis or degradation rate of a protein would likely involve perturbation of specific control mechanisms and not the availability of ATP for polypeptide formation. Likewise, moderate decreases in the concen- trations of several amino acids do not affect protein synthetic rates. Of those amino acids affected, those whose metabolism are closely associated with the tri- carboxylic acid cycle (alanine, glutamate, glutamine, 118 119 aspartate) are most appreciably affected. Changes in concentration of these amino acids may be in response to the depressed energy reserves status and glycolytic rate of the galactose-fed chick. The half-lives of brain protein in the galactose intoxicated chick, measured by the loss of [14C-guanidino] arginine, are not affected over the time period studied. An average half-life of 36 hours was calculated for total protein which is shorter by nearly an order of magnitude than those determined in an adult brain of other species (Chapter II, Discussion). Elevated ammonia levels, a commonly used indicator for increased protein or amino acid breakdown, was not observed in the galactose-treated animals. The stability of neural lysosomes in the galactose- fed chicks to hypoosmotic solutions and incubation at high temperatures is decreased. The increased lability to hypoosmotic shock can be duplicated by preincubation of normal lysosomes in solutions of galactose and galactitol. In addition, the observed increases in the extra-lysosomal activities of B-galactosidase and B-N-acetylhexosaminidase correlate with the summation of the 1g_y1yg concentrations of galactose and galactitol. These data strongly suggest that uptake of galactose and galactitol by the lysosomes leads to increased fragility. Contrary to the carbohy- drase enzymes, acid phosphatase levels did not increase 120 in the cytOplasm consistent with the data of other workers (Chapter IV, Discussion) that there is a differential binding of the acid hydrolases to the lysosomal matrix. It cannot be concluded unequivically that increased lyso- somal enzyme activities in the 20,000 or 100,000 g super- natant preparations are a result of release of enzymes 12_y1yg. Homogenation of the tissue may cause some artificial lysis of the more fragile lysosomes. Radioactively labelled glucosamine and_mannosamine were both incorporated into brain glycoprotein and ganglio- sides at significantly greater rates in galactose-fed chicks than in controls. Although [3H] glucosamine uptake for muc0polysaccharide biosynthesis was also enhanced, normal incorporation rates of 35804 into chondroitin sulfate implies that this subclass of the acid polysaccharides may not be affected. Levels of bound sialic acid and hexosamine did not appear to be significantly altered in any of these fractions. These observations have led to formulation of a hypothesis of increased turnover rate of carbohydrate units on glycoprotein and gangliosides in galactose intoxicated chicks. The possibility of lysosomal dys- function as a result of increased lability or release of acid hydrolases into the cytOplasm is consistent with this hypothesis. In view of the presently held opinions on the central roles played by carbohydrate moieties of 121 glycoprotein and gangliosides in intercellular communi- cation, aberrant metabolism of the carbohydrate residues in plasma or nerve ending membranes could disrupt normal development of neural pathways and hence, neural function. "'ili'lifl‘lfiliijiluifliiujiiiii“WWW” 58 2047