3-Hydroxybutyrate dehydrogenase in tissues from normal and ketonaemic sheep

1. In liver, rumen epithelium and kidney cortex of the sheep, a dehydrogenase active against dl-3-hydroxybutyrate occurred in both the cytosol and particulate fractions of the tissues. In brain, heart, skeletal and smooth muscles, the enzyme occurred only in the particulate fraction. 2. Enzyme activity in the cytoplasmic fraction of liver and rumen epithelium was similar with either d(-)-3-hydroxybutyrate or dl-3-hydroxbutyrate, but was less with acetoacetate as the substrate. The cytosol fraction of kidney cortex showed very little activity with d(-)-3-hydroxybutyrate, confirming that most of the activity with dl-3-hydroxybutyrate was with the l(+) isomer in this tissue. 3. 3-Hydroxybutyrate dehydrogenase activities in the cytosol and particulate fractions of liver, rumen epithelium and kidney cortex and in the particulate fraction of brain tissue were not stimulated by phosphatidylcholine, unlike the enzyme in sheep muscle and in tissues of other species. 4. The activity of 3-hydroxybutyrate dehydrogenase was not increased significantly in any of the tissues of ketonaemic sheep. 5. Comparison of rates of 3-hydroxybutyrate production in vivo with the enzyme activity in ketogenic tissue suggested that in sheep the maximum rate of production might be limited by this activity.     

Differential Activities of Protein Phosphatase Types 1 and 2A in Cytosolic and Participate Fractions from Rat Forebrain

Abstract: The activities and concentrations of protein phosphates type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.     

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.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Modulation of rat myometrial guanylate cyclase activities by sodium nitroprusside and unsaturated fatty acids.

Guanylate cyclase activities are present in both soluble and particulate fractions of rat myometrial extract. Triton, slightly stimulated the soluble (50%) while markedly increasing (1000%) the particulate activity. Both fractions appear to be regulated independently. Predominantly, the soluble form was activated by sodium nitroprusside, involving interactions with SH-groups. On the other hand, the particulate form was stimulated by a series of unsaturated fatty acids and their hydroperoxides. The latter activation appears to result from direct hydrophobic effects rather than peroxide or free radical generation.     

Lysosomal beta-glucosidase of rat liver

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic beta-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-beta-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE DISTRIBUTION OF GLUTAMATE DECARBOXYLASE IN RAT TISSUES; ISOTOPIC VS FLUORIMETRIC ASSAYS

—The activity of glutamate decarboxylase (GAD, EC 4.1.1.15) in normal and neoplastic rat tissues was determined by two assay methods, one based on the production of ¹⁴CO₂ from [¹⁴C]glutamic acid and the other on the fluorimetric measurement of γ-aminobutyric acid (GABA) formation. Activities obtained with the isotopic assay were high in every tissue (ranging from over 800 in liver and brain to 107nmol CO₂/min/g in lung). They were drastically diminished by Triton X-100, by an oxygen-free atmosphere or by the mitochondrial electron transport inhibitors, rotenone and antimycin A. Activities measured fluorimetrically were significant in only a few tissues and were stimulated by Triton (e.g. from 299 to 569 nmol GABA/min/g brain) but were unaffected by rotenone. For several tissues after Triton treatment the fluorimetric and isotopic assays (in air) gave the same results (i.e. the two end products, CO₂ and GABA were in stoichiometric agreement); however, the fluorimetric assay remains the more reliable measure of GAD activity since Triton may not inhibit completely the non-GAD dependent decarboxylation of glutamate in all types of tissue preparations. The hepatic, renal and mammary tumours tested were devoid of GAD; among non-neural normal tissues, kidney, liver and, possibly, adrenal gland contained significant GAD activity. In kidney and liver the activity was 15 and 10 per cent of that in brain.     

Differences in properties of cytosol and membrane-derived protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase present in membrane fractions of bovine brain, heart, liver, and muscle was solubilized with Triton X-100. Certain properties of the membrane-derived enzyme were compared with those of two adenosine 3':5'-monophosphate-dependent protein kinases present in the cytosol fractions from each of the same tissues. The properties studied included chromatographic behavior on DEAE-cellulose columns, specificity with respect to substrate proteins, and sensitivity to NaCl and Triton X-100. The membrane-derived enzyme from each tissue had properties similar to those of the membrane-derived enzyme from each of the other tissues. Moreover, the cytosol enzymes from each tissue had properties similar to those of the corresponding enzymes in the cytosol from each of the other tissues. However, for any given tissue, the properties of the membrane-derived enzyme differed from those of the cytosol enzymes, possibly reflecting different functional roles for the membrane-bound and cytosol enzymes.     

Age-related changes in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the subcellular fractions from the rat brain and the effect of dimethylaminoethanol

The activity of pentose phosphate pathway enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was measured in the cytosol and the particulate fractions (mitochondrial-synaptosomal and microsomal) from the cerebrum and the cerebellum of the rats aged 1, 2, 3, 6, 9 and 12 months. The results showed that the two enzymes occurred both in cytosol and particulate fractions. Both the enzymes were higher in the particulate fractions from cerebellum than in the same fractions from cerebrum. In both regions of the brain, particulate fraction enzymes showed an age-related decline in their activity, but the cytosol fraction enzymes remained unchanged in all the age groups. Dimethylaminoethanol, an important molecular constituent of some antiageing drugs, increased the activity of these enzymes in a dose dependent manner only in the particulate fractions.     

Early activation and redistribution of calpain activity in skeletal muscle during hindlimb unweighting and reweighting

The aims of this study were the following: (i) to determine whether activation of the Ca2+-activated protease, calpain, is an early event during hindlimb unweighting (HU) in skeletal muscle; and (ii) to assess whether calpain activity is greater during reweighting compared with HU alone. Rats were exposed to 12, 24, and 72 h, or 9 d of HU, followed by reweighting for 0, 12, or 24 h. Calpain activities were assayed for total, soluble, and particulate fractions. Total calpain activity was increased in the soleus at all HU time points, whereas activities were elevated in the gastrocnemius only after 9 d of HU. With reweighting, calpain activity remained elevated at all time points for both muscles. In general, reweighting the gastrocnemius increased its calpain activity more than during HU only, whereas reweighting the soleus did not produce additional increases in its calpain activity. The increases in calpain activity were associated with a proportional increase in activity of the particulate (membrane- and protein-associated) fraction. The results suggest that calpain activation is an early event during HU in the soleus, and that the increases in calpain activity in both muscles are associated with a redistribution of activity from cytosolic to particulate fractions.     

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.     

Effect of phorbol esters on the distribution and total activity of protein kinase C in the perfused rat heart

1. The perfused rat heart was treated with the tumour-promoter and protein kinase C activator, phorbol 12-myristate 13-acetate and the distribution of protein kinase C activity between cytosolic and particulate fractions determined. 2. Phorbol ester treatment led to a rapid loss of protein kinase C activity from the cytosol (t0.5 = 2 min) with a corresponding translocation into the particulate fraction. Translocated protein kinase C activity was tightly bound to the particulate fraction, could only be extracted with buffers containing 2% Triton X-100 and could therefore be misinterpreted as being down-regulated. 3. Claims of rapid down-regulation of protein kinase C activity by phorbol esters need to be supported by rigorous procedures for extraction of the particulate material.     

Rapid Translocation of Cytosolic Ca2+/Calmodulin-Dependent Protein Kinase II into Postsynaptic Density After Decapitation

Abstract: The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), α subunit of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to ∼10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X-100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0°C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37°C, by which the content of CaMKIIα in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII α subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKIIβ/β'.     

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.     

The nature of the cytosolic activators of the adrenal steroid 21-hydroxylation system

The steroid 21-hydroxylase activity present in the microsomes of bovine adrenals is stimulated by components of the cytosol. The nature of these activators has been examined by two procedures. The first consisted of treating cytosol with increasing amounts of acetone. When the concentration of the organic solvent reached 50%, a precipitate, presumably proteinaceous, formed. The portion of the precipitate that was redissolvable in 0.05 m potassium phosphate buffer, pH 7.2, contained 7–15% of the stimulatory activity originally present in the cytosol. When the acetone concentration was raised to 90%, another active material precipitated. It was identified as oxidized glutathione (GSSG) and it accounted for about 5% of the activity in the cytosol. In an attempt to avoid the harmful effects of acetone, the second procedure employed only gel filtration and ion exchange resin chromatography. By these means the cytosol was separated into 11 protein fractions and a small molecular weight material. Forty six percent of the proteins and the same fraction of the stimulatory activity present in the original cytosol were recovered. Because all 11 protein fractions contained some stimulatory activity, the results suggested that the protein constituents of these fractions were relatively nonspecific. Yet, of the several known proteins which were tested for activity (bovine serum albumin, ovalbumin, human γ-globulin, bovine pancreatic ribonuclease, and pig insulin) only bovine serum albumin proved to be active. An additional 8% of the stimulatory activity of the cytosol was present in the fraction containing the low molecular weight components and this was all attributable to its GSSG content.     

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.     

Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes.

Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83% of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changed as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83% to 47% of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.