Effects of detergents on Ca2+-activated neural proteinase activity (calpain) in neural and non-neural tissue: A comparative study

Calcium activated neutral proteinase (mcalpain) activity was determined in brain and other tissue of rat. More than 60% of the brain mcalpain activity was present in the particulate fraction while only 30% was in cytosol. In contrast, particulate fractions of liver, kidney, muscle, and heart contained about 8–12% of tissue mcalpain activity while 88% was present in cytosol. Removal of the endogenous inhibitor calpastatin increased the tissue mcalpain activity severalfold. Triton X-100 and deoxycholate (DOC) stimulated the neural calpain activity by ten-fold while activity in non-neural tissue was unaffected. Incubation with other detergents, e.g. Triton N-57 and thioglucopyranoside, stimulated brain calpain activity five-fold while Brij-35 did not have any effect. Sodiumdodecylsulphate (SDS), on the other hand, inhibited the enzyme activity. Brain contained the lowest calpain activity compared to non-neural tissue. The calpain activity in muscle, kidney and heart was three-fold greater than liver. Immunoblot identification of the enzyme revealed that calpain was predominantly in the particulate fraction and less in cytosol of brain while it was present mainly in cytosol and less in the pellet fractions of non-neural tissue.     

Age Peculiarities of the Calpain/Calpastatin Cerebral System in Rats: Relation to the Hypothesis of Brain Aging

We studied age-related changes in the activity of calpain, those in the activity of its endogenous inhibitor calpastatin, and the ratio of these indices in the brain of rats of four age groups (2-3 weeks, 2-3, 5-6, and 24 months). The activity of calpain was estimated using FITC casein as the substrate. In a soluble fraction of the brain homogenate, the enzyme activity in general increased with age. In mature rats (5 to 6 months old), this index exceeded 3.65 times the corresponding index in juvenile (2 to 3 weeks old) animals, while in old animals this index somewhat decreased. In the fraction obtained after separation of calpain from other components using DEAE-cellulose chromatography, the age-related trend toward an increase in the activity of calpain was preserved, but it was much more moderate. The activity of calpastatin demonstrated an opposite direction of age changes: it was the maximum in 2-3-week-old animals and gradually decreased (by 27%) in old rats. We also found that the efficacy of inhibitory action of calpastatin in the cerebral tissue with respect to the activity of calpain was, as a rule, redundant. In this case, the ratio of inhibitor/enzyme activities decreased with age; this index was 1.65, 1.33, 1.1, and 1.0 or less in 2-3-week-old, 2-month-old, mature, and old animals. Therefore, we found that the intensity of calpain-mediated proteolysis in the rat brain increases from the juvenile period to the mature age and somewhat decreases in old individuals. Such alterations are developed at the expense of both an increase in the activity of the enzyme and weakening of the action of its inhibitor (calpastatin).     

Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.     

Degradation of Alzheimer's ß-Amyloid Protein by Human and Rat Brain Peptidases: Involvement of Insulin-Degrading Enzyme

We examined the degradation of Alzheimer's ß-amyloid protein (1–40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the ß-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble ß-amyloid degrading enzyme at neutral pH in human brain. The highest ß-amyloid protein degrading activity in the soluble fractions occurred between pH 4–5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower ß-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases –24.11, –24.15, –24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.     

Soluble protein kinase fractions from DEAF-cellulose chromatography

Summary The cytosol fraction from rat midbrain was chromatographed on DEAE-cellulose with a linear NaCl gradient (0–0.3 M). Two peaks of protein kinase activity were obtained when assayed with either historic or casein. A similar elution profile of the kinase activity was obtained from rat heart. The first peaks from midbrain and heart were compared in terms of their dependency upon cAMP and sensitivity to the endogenous protein kinase inhibitor. Neither of the two substances had an effect on the activity of the brain kinase. Furthermore, the dissociability of the midbrain and heart enzymes in the presence of cAMP or histone was compared by DEAE-cellulose chromatography. The heart enzyme was dissociated into a catalytic subunit characteristic of a cAMP-dependent protein kinase, whereas the brain kinase was totally unaffected by the cAMP or histone. The results of these tests indicate that although the elution profiles from DEAE-cellulose are similar between midbrain and heart, the first peak from brain contains a protein kinase that appears to be cAMP independent.     

Complex Interactions Between Polyamines and Calpain-Mediated Proteolysis in Rat Brain

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high-molecular-weight structural proteins, apparently by activating calcium-dependent proteases (calpains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium-dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine greater than spermidine greater than putrescine, with apparent affinities of 30, 300, and 6,000 microM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium-dependent breakdown of two endogenous high-molecular-weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium-dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a role for polyamines in protein degradation as well as protein synthesis.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Caspase Inhibition Protects Nerve Terminals from in Vitro Degradation

Caspase activation and apoptotic events may take place in terminal regions far removed from the cell body and contribute to synapse loss in neurodegenerative diseases. For examination of events in terminals, we have developed a cell-free assay using quantitative flow cytometric analysis (fluorescence-activated cell sorting) of neuronal particles in a P2 synaptosomal preparation (P-2) from rat brain as a model system. Staurosporine-induced loss of neuronal particles was blocked by nonselective caspase inhibition (z-VAD-fmk) and by calpain inhibition (calpain inhibitor II [ALLM]). Phosphatidylserine exposure was increased in the P-2 by staurosporine treatment, and this increase was blocked by a peptide inhibitor of caspase-3–like activity (Ac-DEVD-CHO). Increased caspase activity in the crude synaptosomal fraction was confirmed by direct measurement with a fluorometric assay. These results indicate activation of both caspase and calpain in the P-2 fraction and suggest a role for these cysteine proteases in the in vitro degradation of nerve terminals.     

Calpain activity in adult and aged human brain regions

We assayed calpain activity in 27 human brain regions from adult (43–65 years of age) and aged (66–83 years of age) postmortem tissue samples. Calpain I (M Ca-requiring) activity was 10% or less of the total activity; it was below detectable levels in a number of areas, and so data are are expressed as total (M+mM Ca-dependent) calpain activity. The distribution of the enzyme was regionally heterogeneous. Highest activity was found in the spinal cord, followed by the amygdala, and levels in mesencephalic areas and in cerebellar grey matter were also high. Levels in cerebellar white matter, tegmentum, pons, and putamen were low, and activity in cortical areas was also relatively low. Although in some areas activity seemed higher with aging, the differences were not statistically significant. We previously found that the regional distribution of cathepsin D in human and in rat brain is similar, this seems to be true for calpain activity as well. The increase of protease activity with age found in rat brain is not found in human areas, as was shown previously with cathepsin D, and in the present study with calpain.Special issue dedicated to Dr. Bernard W. Agranoff.     

Acetylcholine synthesis in human CSF: Implications for study of central cholinergic metabolism

Investigation of neurological diseases involving central cholinergic dysfunction has led to numerous studies seeking a peripheral marker of cholinergic activity in brain. The main objective of these studies was to determine whether the ACh synthesizing activity present in human CSF was due to the presence of the enzyme choline acetyltransferase (ChAT; 68kDa). When CSF was fractionated into low and high molecular weight (M_r) components, 80% of the ACh synthesizing activity (AChSA) was found to be associated with the fraction <10 kDa. The remaining 20% was evenly distributed among fractions in the 5–30, 30–50, 50–300, and 300 kDa fractions. Although boiling destroyed all activity >10 kDa, the ChAT inhibitor NVP, at concentrations equal to or greater than that required to inhibit ChAT in human cortical tissue, did not alter the ACh-SA in either fraction. Results indicate that normal human CSF does not contain ChAT and all ACh-SA in CSF reflects non-enzymatic imidazole/histidine-like catalyzed synthesis.     

Effect of Lipids on the Activity of Calpain in Subcellular Fractions Obtained from the Rat Brain

We studied the effect of lipids on the activity of a neutral cysteine proteinase, calpain, in subcellular fractions obtained from the rat brain. Extraction of nearly 23% of membrane cholesterol from the coarse mitochondrial fraction did not result in modifications of specific activity of calpain in this fraction. Detergents (digitonin or Triton Х-100) used in 0.3% concentration enhanced the activity of calpain in the coarse mitochondrial fraction. Examination of the effects of preparations of different phospholipids on the activity of calpain in the cytoplasm demonstrated that only phosphatidylcholine, but not phosphatidylserine and/or cardiolipin, insignificantly increased the activity of calpain (independently of the size and structure of phospholipid vesicles). We hypothesize that the mechanisms underlying interaction between calpain and lipids are not universal; in native cells and model experiments, they can differ noticeably from each other and are modified depending on the corresponding conditions. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 3–9, January–February, 2009.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

On the distribution of aldolase isoenzymes in subcellular fractions from rat brain

As an extension of previous studies on the adsorption of aldolase (EC 4.1.2.13) in nervous tissue, the main features of the subcellular localization of this enzyme in rat brain have been investigated. The major portion of the aldolase activity in homogenates of this tissue was demonstrated to be present in association with the particulate material, and a differential distribution of the AC isoenzymes was evident between the membranes and the cytosol. Some of the enzyme which was associated with the particulate fraction was shown to be occluded rather than absorbed to the membranes. This type of association was evident in the nuclear and mitochondrial fractions, in particular, with the occluded enzyme presenting an isoenzyme content high in C-type activity, and similar to that of the cytosol. The microsomal fraction contained a high proportion of enzyme in the bound form. Isoenzyme analysis of the enzyme in this microsomal fraction revealed a preferential association between the particulate material and A-type aldolase activity. A purified membrane fraction was prepared from the primary microsomal fraction, and identified as the main site of aldolase binding. The significance of the differential binding of aldolase isoenzymes and its localization amongst the subcellular fractions of rat brain have been discussed in relation to the structural and metabolic features of this tissue, and the coupling of energy producing sequences with energy requiring processes.     

Activation of tyrosine hydroxylase by Ca2+-dependent neutral protease, calpain

Two molecular species of Ca2+-dependent neutral protease (calpains I and II) and its endogenous inhibitor (calpastatin) in cytosol fraction of bovine adrenal medulla were separated by hydrophobic interaction chromatography. Both calpains I and II, having low and high Ca2+ requirements for casein hydrolysis, respectively, were found to activate tyrosine hydroxylase(TH) that had been purified from cytosol fraction of bovine adrenal medulla. This activation of TH by calpain was inhibited by leupeptin and the endogenous inhibitor, calpastatin. The activated TH with calpain II, characterized by high-performance gel permeation chromatography, had a reduced Mr of 120,000 from the Mr of 230,000 of native enzyme.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Developmental changes of calpain and calpastatin in rabbit brain

A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10–20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.