Development of a radioimmunoassay for quantitation of calregulin in bovine tissues

Experimental conditions are described for a convenient and simple one-step method for radioimmunoassay (RIA) of the calcium binding protein calregulin [Waisman, D.M., Salimath, B.P., & Anderson, M.J. (1985) J. Biol. Chem. 260, 1652-1660]. The radioimmunoassay utilizes 125I-labeled calregulin and pansorbin cells (Staphylococcus aureus) coated with rabbit anti-calregulin antibody. Binding equilibrium is attained in 30 min, and the total time of the assay is 1 h. The assay is sensitive to about 30 fmol of calregulin. Calregulin was quantitated in various bovine tissue extracts and was detected in all tissues except erythrocytes. It was present in particularly high amounts in pancreas (540 micrograms/g of tissue), liver (375 micrograms/g of tissue), and testis (256 micrograms/g of tissue). While about 80% of the total tissue calregulin is soluble, about 20% of this protein was found to be associated with particulate fractions. Unmasking of particulate calregulin required the presence of nonionic detergent. Gel permeation chromatography of bovine brain 100 000 g supernatant in the presence or absence of calcium has resolved a single peak of calregulin by RIA. In addition, the distribution of calregulin in the soluble or particulate fraction of bovine brain was unaffected by the presence or absence of calcium during homogenization, suggesting that calregulin does not form either calcium-dependent or calcium-independent association with soluble or particulate proteins. These results identify calregulin as a major tissue Ca2+ binding protein.     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the 'microsomes' (13000-80000 X g sediment) from pea stem tissue is strongly influenced by concentration of Mg2+ in the homogenization medium. The absence of Mg2+ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at 80000 X g upon addition of 5-10 mM Mg2+ (or Mn2+, Ca2+, Zn2+) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl2 the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg2+. Microsomes prepared with Mg2+ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113-0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

Soluble and Participate Forms of the Organophosphorus Neuropathy Target Esterase in Hen Sciatic Nerve

Neuropathy target esterase (NTE) is the suggested "target" molecule involved in the initiation of organophosphorus-induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P-NTE) and soluble (S-NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80-100 nmol/min/g tissue from which 50-60% was recovered in the soluble supernatant fraction and the remaining 40-50% in the pellet fraction. About 90% of brain tissue activity (approximately 1,800 nmol/min/g tissue) was recovered as P-NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P-NTE and S-NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose-6-phosphatase, Na+,K(+)-ATPase, 5'-nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P-NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S-NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane-bound portions of a single protein, with P-NTE activity depending on a membrane factor from which it is separated through fractionation at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Soluble and Particulate Organophosphorus Neuropathy Target Esterase in Brain and Sciatic Nerve of the Hen, Cat, Rat, and Chick

Considerable evidence exists suggesting that the so-called neuropathy target esterase (NTE) is involved in the mechanisms responsible for organophosphorus-induced delayed polyneuropathy (OPIDP). Earlier studies in the adult hen, the habitually employed experimental model in OPIDP, have shown that most NTE activity in the brain is centered in paniculate fractions, whereas approximately 50% of this activity in the sciatic nerve is encountered in soluble form, with the rest being paniculate NTE. In the present work, we have studied the paniculate and soluble fractional distribution of paraoxon-resistant phenylvalerate esterase activity (B activity), parabxon- and mipafox-resistant phenylvalerate esterase activity (C activity), and NTE activity (B - C) according to ultracentrifugation criteria (100,000 g for 1 h). To this effect, two sensitive (adult hen and cat) and two scarcely sensitive (rat and chick) models were used. In all four experimental models, the distribution pattern was qualitatively similar: B activity and total NTE were much greater in brain (900–2, 300 nmol/min/g of tissue) than in sciatic nerve (50–100 nmol/min/g of tissue). The proportion of soluble NTE in brain was very low (<2%), whereas its presence in sciatic nerve was substantial (30–50%). The NTE/B ratio in brain was high for the particulate fraction (>60%) and low in the soluble fraction (7–30%); in sciatic nerve the ratio was about 50% in both fractions. Slight quantitative differences were observed in terms of OPIDP sensitivity: the proportion of soluble NTE in sciatic nerve was slightly higher in the sensitive animals (hen and cat: 49 and 44%, respectively) than in the rat and chick (41 and 37%, respectively), although no differences were noted in terms of concentration (in nanomoles per minute per gram of tissue). It is concluded that the distribution pattern of the activities studied is similar in all four experimental models, with no important quantitative differences directly related to species sensitivity or age.     

A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

Enzymic dephosphorylation of D-myo-inositol 1,4-bisphosphate in rat brain.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.     

Guanine deaminase in rat liver and mouse liver and brain

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis-Menten kinetics. The K(m) value of enzyme A for guanine was 5.3mum and that of enzyme B 20mum. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the K(m) values for the reaction with guanine were respectively 5 and 66mum. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.     

Characterization of arginase activity from mouse epidermis and its relation to ornithine decarboxylase induction by the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate

Arginase, which catalyzes the cleavage of l-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 × g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn²⁺ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55°C in the presence of 15 mM MnCl₂. Enzyme activity in both fractions, in the absence of 15 mM MnCl₂, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar K_m values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12-O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.     

HYPOXANTHINE-GUANINE-PHOSPHORIBOSYL-TRANSFERASE FROM RAT BRAIN (PURIFICATION, KINETIC PROPERTIES, DEVELOPMENT AND DISTRIBUTION)

Hypoxanthine-guanine-phosphoribosyltransferase (HGPR Tase; ECC 2.4.2.8) has been purified from rat brain 650-fold to about 50 per cent purity by conventional methods. An isoenzyme pattern of at least three components is observed on DEAE-cellulose chromatography. On polyacrylamide disc electrophoresis only one sharp band of enzyme activity can be detected. The apparent K_m-value determined for phosphoribosylpyrophosphate (PRPP) is about 0.2 mM. The product, GMP, and also GDP, GTP, UMP, CMP, AMP and ATP are competitive inhibitors with respect to PRPP. Inhibition by a number of other nucleotides has also been investigated. Studies on the development of enzyme activity in the brain of the young rat show that a rapid increase occurs during the first 15-20 days of life and reaches a plateau thereafter. The regional distribution of HGPRTase activity in adult rat brain is more homogenous than that reported for human brain. The enzyme is predominantly a constituent of the soluble supernatant fraction, but can also be found in carefully washed synaptosomes. An antiserum against rat brain HGPRTase obtained from rabbits inhibits this enzyme to about 30 per cent of control activity, but does not crossreact with HGPRTases from rabbit or human erythrocytes.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Protein kinase activity in rat testis interstitial tissue. Effect of luteinizing hormone and other factors.

Protein kinase activity was determined in subcellular fractions of rat testis interstitial tissue after incubation of the intact tissue with LH (luteinizing hormone) in vitro. Various factors that might have changed the activity of this enzyme during preparation of the fractions before assay were also investigated. The following results were obtained. 1. LH and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) added together during incubation of the interstitial tissue caused a twofold increase in the protein kinase activity in the total tissue homogenate and subcellular fractions (12000g X 5 min pellet and 105000g X 60 min supernatant and pellet). 2. A decrease of approx. 40% in the total amount of protein kinase recovered in the soluble fraction (105000g supernatant) occurred in tissue incubated with LH and 3-isobutyl-1-methylxanthine when compared with the controls. No change in total activity was found in the other fractions. 3. LH and 3-isobutyl-1-methylxanthine caused an increase in cyclic AMP concentration in the soluble fraction (from 30 +/- 6 to 450 +/- 40 pmol/mg of protein, means +/- S.E.M., n = 4), but there was little or no increase in the particulate fractions [from 9 +/- 1 to 13 +/- 3 pmol/mg of protein (n = 3) and from 6 +/- 2 to 23 +/- 11 pmol/mg of protein (n = 3) in the 12000g and 105000g pellets respectively]. 4 Addition of 3-isobutyl-1-methylxanthine alone had little effect on protein kinase activity or cyclic AMP concentrations. 5. Little or no protein kinase activity could be demonstrated in subcellular particulate fractions unless Triton X-100 was added; the effect of this detergent was shown to be at least partly due to the inhibition of adenosine triphosphatase activity. 6. In the presence of Triton X-100 approx. 57% of the total protein kinase activity in the homogenate was found in the 105000g supernatant compared with 11% in the 105000g pellet and 32% in the 12000g pellet. 7. In contrast with adipose-tissue protein kinase [Corbin et al. (1973) J. Biol. Chem. 248, 1813-1821] the relative amounts of cyclic AMP-dependent and -dependent enzyme were not affected by dilution of the interstitial-tissue fractions. NaCl (0.5 M) decreased the estimated total amount of protein kinase activity.