Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

Inhibition of sialyltransferase activity in cultured cells by a natural inhibitor from rat brain

Summary Brain from mature rats has been shown previously to contain a natural inhibitor of rat brain sialyltransferase I (CMP-sialic acid: lactosylceramide sialyltransferase activity). This same inhibitor preparation was effective against sialyltransferase I and a second sialyltransferase activity from different lines of cultured cells. Cardiolipin which stimulates sialyltransferase I activity in cultured cells apparently was not required for inhibition. Inhibition was specific for sialyltransferase activities while a third ganglioside biosynthetic enzyme, UDP-gal: glucosyl-ceramide galactosyltransferase activity, was not inhibited. Inhibition of sialyltransferase I was linear with time, heat-resistant, and increased with inhibitor concentration.Gangliosides are sialic-acid containing glycosphingolipids found in brain as well as extraneural tissues and cultured cells t,2. Although the functions of gangliosides are unknown, they appear to play a role in morphological differentiation³ sensory and visual stimulation 4 and as receptor for cholera toxin^5–8 and possibly thyroid stimulating hormone⁹. The monosaccharide units are added to the elongating oligosaccharide chain of the gangliosides in step-wise fashion and different glycosyltransferases catalyze each addition^10,11. The activities of these enzymes have been observed to change during development^12,14 malignant transformation¹³ and morphological differentiation³.Although little is known about the regulation of ganglioside synthesis, a natural inhibitor of CMP-AcNeu: GL-2 sialyltransferase (sialyltransferase I) from rat brain has been described^14,15. As the inhibitor activity increased with the age of the animal, the same authors suggested that it may regulate the biosynthetic pathway of gangliosides. In this paper, the effects of the rat brain inhibitor on the activities of ganglioside biosynthetic enzymes from several cultured cell lines are described.Abbreviations AcNeu N-acetylneuraminic acid - GL1 glucosylceramide - GL-2 lactosylceramide - G_M3 Nacetylneuraminylgalactosylglucosylceramide - G_M1 gal actosyl-N-acetylgalactosaminyl-(N-acetylneuraminylgalactosylglucosylceramide - G_D1a Nacetylneuraminylgalactosyl-N-acetylgalactosaminyl-(Nacetylneuraminyl)-galactosylglucosylceramide. DR. DUFFARD was a recipient of a Fogarty Center Fellowship.     

Identification of bovine brain calcium binding proteins

Three peaks of calcium binding activity have been identified by the Chelex-100 calcium binding assay of the fractions from DEAE cellulose chromatography of 100,000 X g supernatant of bovine brain. These calcium binding activity peaks have been subjected to extensive purification and three novel calcium binding proteins (Mr 27,000, Mr 48,000 and Mr 63,000) and two previously characterized proteins (calcineurin and calmodulin) have been identified as components of calcium binding activity peaks. Analysis of the calcium binding properties of the novel proteins by equilibrium dialysis suggests these proteins may be intracellular calcium receptors.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.     

Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain.

A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.     

Presence of gamma glutamyl transferase in Mycobacterium smegmatis

The presence of gamma glutamyl transferase (GGT) has been established in Mycobacterium smegmatis. The 10,000 x g supernatant demonstrated only hydrolase activity and did not exhibit any transpeptidase activity. Most of the transferase activity was recovered in 100,000 x g supernatant demonstrating that GGT is a cytosolic enzyme. Maximum activity of GGT was observed at two days of growth and the activity decreased significantly till the seventh day of growth when mycobacteria was grown as stationary culture. The km for gamma glutamyl-p-nitroanilide was found to be 0.074 mM and Vmax for the reaction approached 11.9 nmol per min per mg protein. L-serine + borate was found to be a competitive inhibitor (Ki 12.05 mM) for GGT activity. The pH optimum for GGT activity was observed between 7.5 to 8.5 and temperature above 35 degrees C rapidly inactivated the enzyme activity. To the best of our knowledge, this is the first report which unequivocally establishes the presence of GGT activity in 10,000 x g supernatant of M. smegmatis.     

OCCURRENCE OF A SEROTONIN SULPHOTRANSFERASE IN THE BRAIN

Abstract— —An enzyme catalysing the transfer of sulphate from 3'-phosphoadenylsulphate to serotonin was purified from rabbit brain. The purification procedure involved ammonium sulphate fractionation of the 200,000 g supernatant of rabbit brain homogenate, treatment with alumina Cγ, and chromatography on DEAE-cellulose. The enzyme was purified 67-fold from the 200,000 g supernatant of the brain homogenate. The intracranial distribution of the sulphotransferase was investigated and the cerebellum found to have rather high activity. The sulphotransferase activities of rabbit, dog, rat and bovine brains were compared; rabbit brain had the highest activity, followed by dog, rat and bovine brain.     

Inhibition of rat heart and brain cyclic adenosine-3',5'-monophosphate phosphodiesterase in vitro under the influence of neurohormone C]

The work was devoted to one of the new neurohormones (neurohormone "C") obtained from the bovine hypothalamus which produced a pronounced relaxation of the coronary vessels. A study of the effect of "C" on cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (PDE) showed "C" to be a potent inhibitor of PDE in the crude PDE-preparations of the rat heart and brain. Experiments were carried out on the 2000 g supernatant obtained from the homogenized tissues. It is supposed that the relaxant effect of "C" on the coronary vessels is due to its inhibitory action on PDE mediated by the accumulated cAMP.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Characterization of ribonucleases and ribonuclease inhibitor in subcellular fractions from rat adrenals

1. The presence of two RNA-degrading enzymes, one with optimum activity at pH5.6 (acid ribonuclease) and the other with optimum activity at pH7.8 (alkaline ribonuclease), in rat adrenals has been demonstrated. The acid ribonuclease was localized in the mitochondrial fraction whereas the alkaline ribonuclease was present in mitochondria as well as in the supernatant fraction. Freezing and thawing of mitochondria and treatment with Triton X-100 gave a three- to four-fold increase in acid-ribonuclease activity, whereas the mitochondrial alkaline-ribonuclease activity was practically unaffected. 2. The amount of free ribonuclease in the adrenal supernatant was small. Treatment of the supernatant fraction with N-ethylmaleimide resulted in release of large amounts of ribonuclease activity, indicating the presence of a ribonuclease inhibitor having reactive thiol groups. 3. Considerable amounts of free ribonuclease inhibitor in excess over the bound alkaline ribonuclease are present in the rat-adrenal supernatant fraction. The inhibitor is heat-labile and non-diffusible. A 400-500-fold purification of the ribonuclease inhibitor was achieved by ammonium sulphate fractionation, treatment with calcium phosphate gel and DEAE-cellulose chromatography. It is concluded that the adrenal inhibitor is protein in nature, similar to the inhibitor present in rat liver.     

Enzymatic oxidation of all-trans retinal to retinoic acid in rat tissues

The 100,000 x g supernatant (cytosolic) fraction of rat tissue homogenates catalyzes the oxidation of all-trans retinal to retinoic acid. Kidney, testis, and lung were the most active of the tissues examined. The presence of enzyme activity in liver and intestine could be detected only when a substrate concentration beyond the saturation point for retinal reductase was used. Spleen, brain, and plasma had no activity. Boiled supernatants did not catalyze the reaction. The enzymatic product was chemically and physically identified as retinoic acid. The cytosol of kidney tissue also catalyzed the conversion of retinol to retinoic acid. These data indicate that kidney tissue has the highest retinal oxidase activity and suggest that it may play a major role in the oxidative metabolism of retinol in the body.     

Cholesterol esterase in rat adipose tissue and its activation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

A high level of cholesterol esterase activity, comparable to that of hormone-sensitive triglyceridase, has been demonstrated in rad adipose tissue. Essentially all of the activity was in the isolated adipocytes, primarily in the 100,000 times g supernatant fraction of the adipocytes. Cholesterol esterase activity in the 100,000 times g supernatant fraction was increased 40 plus or minus 16% by incubation with ATP (0.5 mM), Mg-2+ (1.25 mM), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) (10 muM), conditions which also activated hormone-sensitive triglyceridase. Protein kinase inhibitor (rabbit skeletal muscle) blocked activation, and activation was restored by the addition of excess protein kinase (bovine skeletal muscle). In extracts prepared from adipocytes first incubated for 5 min with 10 muM epinephrine and 1 mM theophylline, there was no cyclic AMP-dependent cholesterol esterase activation, implying that the enzyme had been activated by a similar mechanism in the intact cell. The physiological role of this high level of cholesterol esterase activity in adipose tissue is unclear. Its relationship to hormone-sensitive triglyceride lipase, with which it extensively co-fractionates, and its possible involvement in fat mobilization remain to be determined.     

Characterisation of a Myristoyl CoA:Glycylpeptide N-Myristoyl Transferase Activity in Rat Brain: Subcellular and Regional Distribution

Abstract: An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60^src. This N -myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Mi-chaelis-Menten kinetics yielding K _m values of 100 μ M and 60 μ M , and V_max values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and sre-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N -myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord.     

Formation of a New Biogenic Aldehyde Adduct by Incubation of Tryptamine with Rat Brain Tissue

Tryptamine was degraded by incubation with rat brain homogenate to an unknown product. The reaction was stimulated by the nonionic detergents Triton X-100 and Lubrol PX and less by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS). The same results were obtained with pig brain and bovine brain. The monoamine oxidase inhibitor pargyline inhibited the reaction strongly, indicating the participation of the enzyme on the reaction. Addition of 17,000 g supernatant from rat brain homogenate increased the formation effectively whereas phospholipids or chloroform/methanol (7:3) extract from the 17,000 g supernatant showed only little or no effect. Chromatographic and electrophoretic properties as well as the chemical reaction of the product with specific reagents suggest that the compound consists of an indole part and an amino acid part. The product could be identified by fast atom bombardment mass spectrometry and by comparison with the synthetic substance (4R)-2-(3-indolylmethyl)-1,3-thiazolidine-4-carboxylic acid. It is formed by the enzymatic oxidation of tryptamine producing indole-3-acetaldehyde which spontaneously cyclizes with free L-cysteine from the tissue. The results suggest that the reaction of biogenic aldehydes with brain macromolecules may proceed via an analogous reaction.     

Solubilization of glucagon and epinephrine sensitive adenylate cyclase from rat liver plasma membranes

Hormonally sensitive adenylate cyclase has been solubilized from rat liver plasma membranes using Triton X-305 in Tris buffers containing mercaptoethanol and MgCl₂. The solubilized enzyme was stimulated 5 fold by NaF, 7 fold by glucagon and 20 fold by epinephrine. Criteria for solubilization included lack of sedimentation at 100,000 × g for one hour, the absence of particulate material in the 100,000 × g supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cyclase activity in Sephadex G 200 gels. The molecular weight of the solubilized, hormonally sensitive enzyme was approximately 200,000 in the presence of Triton X-305.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Squalene-2(3)-epoxide-lanosterol cyclase in developing rat brain

Abstract— Squalene-2(3)-epoxide-lanosterol cyclase activity has been studied in cell-free extracts of developing rat brain. The enzyme is microsomal and is not stimulated by the addition of 100,000 g supernatant fluid. It is activated by deoxycholate in a manner similar to that of the liver cyclase. It is not inhibited by nicotinamide over a wide range of concentrations but is strongly inhibited by the hypocholesteraemic agent N-dodecylimidazole. Cyclase activity increases from low levels at 5 days of age to maximum activity at 13 days and thereafter slowly declines. The increase in activity is independent of increases in total brain protein and has been analysed by the method of ‘chemical allometry’. Using this technique it can be seen that prior to 13 days the proportion of brain protein allotted to cyclase is increasing whereas after this time it decreases.     

A protein kinase inhibitor in brown adipose tissue of developing rats

A heat-stable protein was extracted from brown adipose tissue of infant rats that inhibited both purified bovine heart protein kinase and a crude preparation of protein kinase from brown fat. It did not act as an adenosine triphosphatase nor did it exert its effect by proteolytic action. Inhibition of protein kinase was affected in both the presence and the absence of cyclic AMP. Most of the inhibitory activity was present in the 100000g supernatant fraction of tissue homogenates. Inhibitory activity was highest perinatally and it declined 10 days after birth. It is suggested that the protein kinase inhibitor may play a role in regulating cyclic AMP actions.