Tartrate-resistant acid phosphatase 5b as a serum marker of bone metastasis in breast cancer patients

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 ± 1.1 U/I, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 ± 0.64 vs. 2.83 ± 1.1 U/I; p=0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 ± 2.5 vs. 2.83 ± 1.1 U/I; p<0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p=0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p<0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.     

A crucial role of interleukin-6 in the pathogenesis of thyrotoxicosis-related disturbances of bone turnover in mice.

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by a negative balance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) reflecting resorption, and osteocalcin as a marker of bone formation in IL-6 knock-out mice to assess the role of IL-6 in the pathogenesis of thyrotoxicosis-related disturbances of bone metabolism. The study was performed on forty, 14-15 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6J (IL6-/-Kopf) (IL-6 knock-out; IL6KO). The mice were randomly divided into 4 groups with 10 mice in each one: 1. WT mice in thyrotoxicosis (WT-thx), 2. WT controls (WT-ctrl), 3. IL6KO mice with thyrotoxicosis (IL6KO-thx), and 4. IL6KO controls. Experimental model of hyperthyroidism was induced by intraperitoneal injection of levothyroxine at a dose of 1 microg/g, daily over 21 days. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentration of TRACP 5b (median and interquartile ranges) were significantly increased in both groups of mice with thyrotoxicosis: WT [28.2(18.8-41.6) U/l] and IL6KO [26.4(23.0-31.2) U/l] as compared to the respective controls. Osteocalcin serum levels in IL6KO-thx mice [111.9(103.1-175.6) ng/ml] were significantly elevated in comparison to WT-thx animals [46.1(32.5-58.9) ng/ml]. In summary, the results of the present study suggest that IL-6 plays a crucial role in thyrotoxicosis-related disturbances of bone turnover in mice, determining the imbalance between bone resorption and bone formation caused by excess of thyroid hormones predominantly by inhibition of bone formation.     

Interleukin-6 is not essential for bone turnover in hypothyroid mice

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by an imbalance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) and osteocalcin in IL-6-deficient mice to assess the role of IL-6 in bone metabolism in hypothyroidism in mice. C57BL/6J (wild-type; WT) and C57BL/6J(IL6-/-Kopf) (IL-6 knock-out; IL6KO) mice randomly divided into 4 groups with 10 in each one: 1/ WT mice in hypothyroidism (WT-ht), 2/ WT controls, 3/ IL6KO mice with hypothyroidism (IL6KO-ht) and 4/ IL6KO controls. Experimental model of hypothyroidism was induced by intraperitoneal injection of propylthiouracyl. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentrations of TRACP 5b (median and interquartile ranges) were significantly decreased in both groups of mice with hypothyroidism: WT (3.2 (2.5-4.7) U/l) and IL6KO (2.6 (1.8-3.5) U/l) as compared to the respective controls. Similarly, serum osteocalcin levels were significantly reduced in both groups of mice in experimental hypothyroidism: WT (25.8 (23.0-28.2) ng/ml) and IL6KO (21.5(19.0-24.6) ng/ml) in comparison to the respective controls. There were no significant differences in bone turnover markers between IL6KO and WT mice both in hypothyroid and control animals. The results of the present study suggest that IL-6 does not play an important role in bone turnover in both euthyroid and hypothyroid mice.     

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.     

Macrophages overexpressing tartrate-resistant acid phosphatase show altered profile of free radical production and enhanced capacity of bacterial killing

Activated macrophages and osteoclasts express high amounts of tartrate-resistant acid phosphatase (TRACP, acp5). TRACP has a binuclear iron center with a redox-active iron that has been shown to catalyze the formation of reactive oxygen species (ROS) by Fenton’s reaction. Previous studies suggest that ROS generated by TRACP may participate in degradation of endocytosed bone matrix products in resorbing osteoclasts and degradation of foreign compounds during antigen presentation in activated macrophages. Here we have compared free radical production in macrophages of TRACP overexpressing (TRACP+) and wild-type (WT) mice. TRACP overexpression increased both ROS levels and superoxide production. Nitric oxide production was increased in activated macrophages of WT mice, but not in TRACP+ mice. Macrophages from TRACP+ mice showed increased capacity of bacterial killing. Recombinant TRACP enzyme was capable of bacterial killing in the presence of hydrogen peroxide. These results suggest that TRACP has an important biological function in immune defense system.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

A protein phosphotyrosine phosphatase distinct from alkaline phosphatase with activity against the insulin receptor

Rat liver plasma membranes were found to have a relatively high ratio of acid to alkaline phosphatase activity when compared to rabbit liver and human placental membranes, respectively. The rat liver plasma membranes contained PPTl phosphatase activity against the soluble autophosphorylated insulin receptor beta-subunit. The PPT phosphatase activity of the membranes, using 32P-histone 2b as a substrate, was inhibited by 100 microM Zn+2, insensitive to 10 mM EDTA, and displayed maximal activity at neutral pH. Dephosphorylation of the insulin receptor beta-subunit by rat liver membranes was inhibited by Zn+2, and stimulated by EDTA. These results prove that the plasma membrane of a physiologically relevant insulin target tissue contains a PPT phosphatase, distinct from alkaline phosphatase, which catalyzes the dephosphorylation of the insulin receptor beta-subunit.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

Background

Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.

Methods

A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP^® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.

Results

The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R² = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).

Conclusions

High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

Subcellular distribution of acid and neutral alpha-glucosidases in normal, acid maltase deficient, and myophosphorylase deficient human skeletal muscle

The subcellular distribution of acid (pH 4.0) and neutral (pH 6.5) α-glucosidases was investigated in biopsy specimens of human skeletal muscle obtained from six normal subjects, four adult cases of acid maltase deficiency, and a case of myophosphorylase deficiency. The highest relative specific activity of acid glucosidase, as well as of other acid hydrolases, was observed in the light mitochondrial fraction. Relatively high acid phosphatase activity was also found in the microsomal fraction. In all muscles the highest relative specific activity of neutral glucosidase was in the microsomal fraction. In acid glucosidase deficient muscle no neutral glucosidase could be detected in the light and heavy mitochondrial fractions but in normal and myophosphorylase deficient muscle neutral glucosidase activity was also detectable in these fractions. The final supernatant of all muscles contained neutral glucoamylase activity. The relevance of these data to the pathogenesis of the different forms of type II glycogenosis is considered.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

Molecular forms of alkaline phosphatase of ram seminal plasma — Some properties and changes in pathological process of reproductive organs

Three molecular forms of alkaline phosphatase were isolated from ram seminal plasma. These forms, activated with Mg²⁺ ions, were characterized by very similar pH optima, K_m constant, and molecular weight. They differed in electrophoretic mobility, the latter being most probably determined by the different position of N-acetylneuraminyl groups in protein structures. Sialic acid also played a protective function for the catalytic centre.Isolated molecular forms possessed antigenic properties. Immunological serum for phosphatase proteins either inhibited or stabilized activity of alkaline phosphatase, depending on the value of the protein ratio.During experimentally induced inflammation of ram reproductive organs, a gradual decrease of the activity of alkaline phosphatase was noted, together with changes in its electrophoretic profile. This phenomenon is most likely caused by intensive synthesis of sialic acid in pathologically changed reproductive organs of the ram.     

A Generic Mechanism of β2-Microglobulin Amyloid Assembly at Neutral pH Involving a Specific Proline Switch

Although numerous measurements of amyloid assembly of different proteins under distinct conditions in vitro have been performed, the molecular mechanisms underlying the specific self-association of proteins into amyloid fibrils remain obscure. Elucidating the nature of the events that initiate amyloid formation remains a particularly difficult challenge because of the heterogeneity and transient nature of the species involved. Here, we have used site-directed mutagenesis to create five proline to glycine variants in the naturally amyloidogenic protein β₂-microglobulin (β₂m). One of these variants, P5G, allowed us to isolate and characterise an intermediate containing a non-native trans Pro32 backbone conformation, a feature that is known to be required for amyloid elongation at neutral pH. By analysing oligomerisation and amyloid formation using analytical size-exclusion chromatography, multi-angle static light-scattering, analytical ultracentrifugation, circular dichroism and thioflavin T fluorescence we reveal a pathway for β₂m amyloid assembly at pH 7.5 that does not require the addition of metal ions, detergents, co-solvents or other co-factors that have been used to facilitate amyloid formation at physiological pH and temperature. Assembly is shown to involve the transient formation of a non-native monomer containing a trans P32 backbone conformation. This is followed by the formation of dimeric species and higher molecular mass oligomers that accumulate before the development of amyloid fibrils. On the basis of these results, we propose a generic mechanism for β₂m fibrillogenesis at neutral pH that is consistent with the wide range of published studies of this protein. In this mechanism, amyloid formation is initiated by a specific cis to trans proline switch, the rate of which we show to be controlled by the amino acid sequence proximal to P32 and to the applied solution conditions.     

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

Isoelectric focusing in immobilized pH gradients: applications in clinical chemistry and forensic analysis

The applications of isoelectric focusing in immobilized pH gradients in clinical chemistry and forensic analysis are reviewed. Strong emphasis is given to the separation of serum proteins, in particular α₁-acidic glycoprotein, acid phosphatase, alkaline phosphatase, α₁-antitrypsin, apolipoproteins, complement component, factor B, factor XIIIB, group-specific component, lecithin:cholesterol acyltransferase, phosphoglucomutase, prealbumin, protein C and transferrin. The analysis of human parotid salivary proteins is discussed and an assessment is given of the state of the art in thalassaemia screening.     

Oxidation of proteins: is it a programmed process?

Proteins represent extremely susceptible targets for oxidants. Oxidative modifications of proteins may bring about violation of their structure and functionality. It implies that the structures of proteins are not infallible in terms of their antioxidant defence. The protection mechanisms in preventing oxidative damages for proteins within cells are mainly related to a large variety of antioxidant enzymatic systems. In contrast, plasma proteins are scarcely protected by these systems, so the mechanism that provides their functioning in the conditions of generating reactive oxygen species (ROS) seems to be much more complicated. Oxidation of many proteins was long considered as a random process. However, the highly site-specific oxidation processes was convincingly demonstrated for some proteins, indicating that protein structure could be adapted to oxidation. According to our hypothesis, some of the structural elements present in proteins are capable of scavenging ROS to protect other protein structures against ROS toxicity. Various antioxidant elements (distinct subdomains, domains, regions, and polypeptide chains) may act as ROS interceptors, thus mitigating the ROS action on functionally crucial amino acid residues of proteins. In the review, the oxidative modifications of certain plasma proteins, such as α₂-macroglobulin, serum human albumin, fibrinogen, and fibrin-stabilising factor, which differ drastically in their spatial structures and functions, are analysed. The arguments that demonstrate the possibility of existing hypothetical antioxidant structures are presented. For the first time, the emphasis is being placed on the programmed mechanism of protein oxidation.     

Discussion of the role of the extracellular signal-regulated kinase-phospholipase A2 pathway in production of reactive oxygen species in Alzheimer's disease

In this paper we show that exposure of a rat brain synaptosome fraction to the amyloid beta peptide fragment A(25-35), but not the inverted peptide A(35-25), stimulated production of reactive oxygen species (ROS) in a concentration- and time-dependent manner. The ROS formation was attenuated by the tyrosine kinase inhibitor genistein, the mitogen-activated protein kinase inhibitor U0126, and the phospholipase A₂ (PLA₂) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid. This strongly suggests that A(25-35) stimulated ROS production through an extracellular signal-regulated kinase-PLA₂-dependent pathway. The interaction between these enzymes and their possible involvement in free radical formation in Alzheimer's disease are discussed.     

The localization of proteolytic activity in rat liver mitochondria and its relation to mitochondrial swelling and aging

1. On storage of rat liver mitochondria at 0°, water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1·9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22° and 40°, the concentration gradient reaching 70μmoles/ml., water content rising to 8·3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. `Swelling agents' produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. β-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was `free' in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was `free'. 4. `Light' mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than `heavy' mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7·4 and 70% at pH5·8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40–60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5·0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.