Julian N. Kanfer

Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction

Abstract Phospholipase D activity (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was demonstrated in vitro in a solubilized preparation from rat brain particulate fraction which also possessed the transphosphatidylation activity. The preparation attacked a phosphatidylcholine microdispersion and cleaved the terminal phosphate diester bond of this phospholipid resulting in the formation of phosphatidic acid. The pH optimum for the phosphatidohydrolase activity was broad with an apparent peak aroung 6.0 whereas the transphosphatidylation showed a sharp pH optimum at 7.2 Ca2+ was not essential for the hydrolysis, but merely stimulated slightly with an optimum about 5 m m , however, it could be replaced by Mg2+. The hydrolysis of phosphatidylcholine by the enzyme was almost completely inhibited in the presence of either diethyl ether (20% by volume) or p-chloromercuriophenyl sulfonate (6 × 10 −5 m ). The latter inhibition was reduced by the addition of dithiothreitol (6 × 10−4 m ). The result suggests an essential role of sulfhydryl group in the formation of the enzyme-substrate complex. The apparent Km for phsophatidylcholine for the phosphatidohydrolase activity was about 8.3 × 10 −4 m .

Solubilization and properties of a membrane-bound enzyme from rat brain catalyzing a base-exchange reaction

Abstract A solubilized base-exchange enzyme activity was dependent upon the addition of phospholipids, added Ca++ ion, and had optimum pH of 7.2. Phosphatidyl-ethanolamine was found to be the best stimulator of both[14C]-ethanolamine and [14C]-serine incorporation. Preliminary evidence suggests the presence of phospholipase D type activity in this solubilized preparation.

Lysosomal enzyme variations in cultured normal skin fibroblasts

Abstract Lysosomal enzyme activities were studied in normal cultured fibroblasts derived from a single skin biopsy, grwon in quintuplet and treated in an identical way. A 60–500% variation in enzyme activities were noted. These observations compound the difficulties of accurate homozygote and heterozygote detection using cultured fibroblasts.

Copper Deficiency and The Central Nervous SystemMyelination in the Rat: Morphological and Biochemical Studies

Less histologically demonstrable myelin was present in the central nervous system of newborn rats having a mean brain copper concentration only 20% that of normal. Tremor, spontaneous tail elevation and a loss of aggressive character were consistent features. Significant decreases in typical myelin lipids, sulfatide, and especially galactocerebroside were found in agreement with the histological deficit. The composition of gangliosides and phospholipids were unaltered except for a slight enrichment in phosphatidylcholine. Galactosylation of ceramide, in vitro, was depressed in copper-deficient brain and the relevance of this finding is discussed. Swayback, a non-febrile, ataxic disorder of newborn and young lambs, was first reported by Bennetts in Western Australia (4, 5). The disease is associated with low levels of copper in tissue of both ewes and affected lambs (6) due to low copper content in pastures. Supplementation with copper of either the ewe during pregnancy, or the lamb directly, prevents manifestations of the disease (7, 15). Although a well known entity in sheep, a condition resembling swayback also has been reported in copper-deficient goats (26, 37, 41) and pigs (33, 50). The common pathological features of swayback include symmetrical demyelination with subsequent cavitation of the cerebral white matter and degeneration of descending tracts in the spinal cord (28, 42, 43); however, myelin aplasia has also been observed (27). Similar changes have been induced in lambs (31) and guinea pigs (16) born to mothers fed experimental diets deficient in copper, but myelin changes have not been reported in the copperdeficient rat. Demyelination and amyelination accompanying swayback disease in sheep are associated with a depletion of typical myelin lipids such as cerebroside (27). The decrease in lipids may be involved in the pathogenesis of the disease in the nervous system, but the basic defect that results in decreased myelin lipid is not known. Recently, DiPaolo and Newberne (13) observed a variety of clinical and morphological abnormalities in copper-deficient newborn rats which include a high mortality, retarded growth, petechinal and ecehymotic hemorrhages in brain, muscle and subcutaneous tissue, focal necrosis in the brain associated with neonatal incoordination of the hind limbs, hemorrhagic necrosis of the liver, cardiac hemorrhage, and severe, widespread vascular dilation in cardiac walls and septae. We also observed behavioral changes in rats having a mean brain copper concentration about 20% of normal. The purpose of this report is to describe further these changes, present the histological myelin anomalies observed in these animals, and correlate this morphological observation with quantitation of brain lipids and the in vitro synthesis of galactocerebroside.

Studies on base-exchange reactions of phospholipids in rat brain particles and a “solubilized” system

Abstract A filtration-method on Millipore-membranes for the assay of the base-exchange reaction was described. Its advantage over the usual procedure based upon the extraction and the washing of lipids was discussed with the viewpoint of processing many samples, which would be indispensable for purifying the enzyme. The reaction showed an absolute dependency for calcium ion with different optimal concentrations for each of the three bases, a sensitivity to inhibition by high ionic strength, and a pH optimum around 9.0. Exogenously added phospholipid, asolectin, gave a slight stimulation for ethanolamine and l -serine incorporation at a low concentration while choline incorporation was essentially inhibited at all concentrations examined. In heat-denaturation experiments with the particulate and soluble the incorporation of choline into lipid was more sensitive than that of ethanolamine and l -serine. A developmental study showed that brain particles sedimenting between 10,000 and 35,000g prepared from rats aged 22–27 days readily incorporated ethanolamine, l -serine, and choline into their corresponding phosphatidyl compound. Several procedures for solubilization of the “base-exchange” enzyme were examined. The most effectively solubilized preparation was obtained by the use of an ionically balanced detergent, Miranol H2M. This preparation showed a marked dependency on exogenously added phospholipids for its maximal enzymic activity, had a pH optimum at around 7.2, and had an absolute requirement for Ca2+. This particular detergent at a concentration of 1% ( w v ) solubilized approximately 50% of the protein, and about 30% of the phospholipids, 40% of the cerebrosides, and only 11% of the cholesterol originally present in the particles. The relative proportions of different phospholipids solubilized by the detergent were, however, similar to those present in the original particles. The base-exchange reaction catalyzed by the solubilized enzyme was found to be highly sensitive to ionic strength, and the inhibitory effect of a specific monovalent cation paralleled its ionic size. Substantial differences in the Km value for each of the substrates with only slight differences in V were observed. The choice of solubilizing agents in relation to these properties and to the maintenance of the activity of the base-exchange reaction was discussed.

On the loss of gangliosides by dialysis

The Gangliosides represent a family of sialic acidcontaining sphingoglycolipids which have ceramide (N‐acyl‐sphingosine) as the basic hydrophobic portion (Svennerholm, 1972). A heteropolysaccharide is glycosidically linked through the primary hydroxyl group of ceramide and the aldehyde of glucose. There are usually three or four different carbohydrates and up to a total of seven in the chain. The isolated gangliosides are soluble both in organic and aqueous solvents presumably because of their content of both hydrophobic and hydrophilic groups. The most commonly employed extraction procedure is based upon their solubility in chloroform‐methanol (2: 1, v/v) and subsequent partitioning into an aqueous methanol phase (FOLCH, LEES and SLOANE‐STANLEY, 1957). This methanolic solution is then dialysed against water to remove small molecular‐weight contaminants. This paper reports the loss of gangliosides upon dialysis when they are present at low concentrations.

Studies on base-exchange reactions of phospholipids in rat brain: Heterogeneity of base-exchange enzymes

Abstract The solubilization of base-exchange enzymes from rat brain was effected with a combination of 0.8% Miranol H2M with 0.5% sodium cholate. The yields of incorporation activities for ethanolamine and serine in the solubilized supernatant fraction were 74 and 65%, respectively. Choline incorporation enzyme was not solubilized by this detergent mixture. The solubilized activities for ethanolamine and serine were concentrated into the fraction precipitated by (NH 4 ) 2 SO 4 between 35 and 55% in the presence of Asolectin dispersion. The separation of base-exchange enzyme specific for each of the three compounds was accomplished with chromatography using Sepharose 4B, Affi-Gel 102 and DEAE-cellulose. Separate fractions were obtained which possessed principally choline or serine or ethanolamine incorporation activities.

L‐DOPA: Effect on cerebral pyridoxal phosphate content and coenzyme activity

L-DOPA is currently being used as therapy for certain of the neurological symptoms associated with Parkinsons disease (BARBEAU, 1969 ; C~TZIAS, PAPAVASILIOU and GELLENE, 1969; YAHR, DIDOISIN, HOEHN, SCHEAR and BARREIT, 1968). This drug is converted by a vitamin B6-dependent decarboxylation reaction to dopamine, which is a neurotransmitter in the basal ganglia. Patients taking L-DOPA are not effectively managed if they are concurrently receiving vitamin B6 (DUVOISIN, YAHR and C~T,TE, 1969). The present report describes studies in vivo to assess certain effects of the administration of L-DOPA on the vitamin B6 coenzyme, pyridoxal phosphate, in the brain. L-DOPA was mixed in 5 ml of an aqueous suspension of 0.25 % (w/v) Keltrol (Xanthan Gum; donated by Kelco, Clark, N.J.) and administered to adult Charles River strain rats by stomach tube in dosages of 125 mg/kg, 1 g/kg and 2.5 g/kg body wt. Control animals received an identical suspension devoid of L-DOPA. The animals were killed by decapitation 24 h later and their brains were removed. To establish the cerebral levels of pyridoxal phosphate, onehalf of the brain from each animal was homogenized in 10 vol. of 0.2 M-perchloric acid and the mixture was centrifuged at 45,000 g for 30 min. The supernatant fluid was removed and adjusted to pH 5.5 with KOH, and the insoluble potassium perchlorate was discarded. The content of vitamin B6 in the resulting supernatant fluid was quantitated with the vitamin B6-dependent tyrosine decarboxylase (L-tyrosine carboxy-lyase; E.C. 4.1.1.25) (Worthington Biochemicals, Freehold, N.J.) (MARUYAMA and COURSIN, 1968). The decarboxylation of [4C]tyrosine by this preparation was proportional to the amount of pyridoxal phosphate in the incubation mixture. The l4COZ evolved was trapped in Hyamine and the radioactivity was counted by liquid scintillation spectrometry. Recovery experiments were carried out with mixtures of known quantities of pyridoxal phosphate and serum albumin. Another vitamin B6-dependent enzyme, glutamate decarboxylase (L-glutamate I-carboxy-lyase ; E.C. 4.1.1.15), was assayed in the following manner. The remaining one-half brain from each animal was homogenized in 5 vol. of HzO, and varying amounts of the suspension were assayed for the activity of the glutamate decarboxylase (EBERLE and Er~uSo~,1968 ; TAPIA, PASANTES and MASSIEU, 1970). The 14C0, evolved from [14C]glutamic acid was trapped in Hyamine and the radioactivity was counted by liquid scintillation spectrometry.

Purification of α- and β-galactosidases by affinity chromatography

Abstract Jack bean meal β-galactosidase and ficin α-galactosidase were purified 48- and 76-fold, respectively, by chromatography on galactonate-benzidine Sepharose. The use of sugar acid lactones coupled to an inert support may provide a generally applicable method for lysosomal acid hydrolase purification.

Acid hydrolases in neuronal and glial enriched fractions of rat brain

Fractions enriched in neuronal and glial cells from rat brain were assayed for several lysosomal acid hydrolases, namely, β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23), β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), α-galactosidase (α-D-galactoside galactohydrolase, EC 3.2.1.22), α-mannosidase (α-D-mannoside mannohydrolase, EC 3.2.1.24), N-acetyl-β-glucosaminidase (β-2-acetamido-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30), N-acetyl-β-galactosaminidase, acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2), β-glucuronidase (β-D-glucuronide glucuronohydrolase, EC 3.2.1.31), arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1), glucocerebrosidase and galactocerebrosidase. The data do not show any significant difference in the distribution of these activities between the two cell types.