The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

CHARACTERIZATION OF A RAT BRAIN CATHEPTIC CARBOXYPEPTIDASE (CATHEPSIN A) INACTIVATING ANGIOTENSIN-II

—Catheptic carboxypeptidase (cathepsin A) is present in lysosomal-enriched fractions of rat brain at levels approximately 5-fold that of cathepsin B1 and of the classical carboxypeptidase A but lower than that of cathepsin C and carboxypeptidase B. Cathepsin A was purified 40-fold by extraction of calf brain with an acetate buffer containing 0.5% desoxycholate followed by heat treatment, salt precipitation and chromatography on DEAE-Sephadex. Purification revealed the presence of two distinct isoenzymatic forms of high mol. wt that were very stable when frozen in the presence of sucrose and KCl. Among N-protected dipeptides used as substrates the highest activity was given by Z-Glu-Tyr and Z-Phe-Tyr at a pH optimum of 5.5, K_m1.1 mm and V_max 0.5 μmol (Tyr)/mg protein per min. Brain cathepsin A was completely inhibited by low concentrations of DFP and PCMB but unaffected by thiols, EDTA and specific inhibitors of other cathepsins (pepstatin and chymostatin). The carboxypeptidase A-like specificity of cathepsin A was confirmed by breakdown of Ile⁵-angiotensin with release of C-terminal Phe. Cathepsin A may play a role in the turnover of selected hormonal peptides containing C-terminal neutral amino acids and in the sequential breakdown of proteins associated with degenerative conditions such as demyelination.     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors

Most previous studies on inhibitors of lysosomal protein breakdown have been performed on isolated or cultured cells or on perfused organs. We have tested various inhibitors of proteolysis on lysosomes isolated from livers of rats injected with [14C]leucine 15 min (short labeling time) and 16 h (long labeling time) before killing. Intact lysosomes were incubated with different inhibitors (leupeptin, propylamine, E-64, pepstatin, and chloroquine) in increasing concentrations. None of these caused more than a 40-75% inhibition of proteolysis irrespective of labeling protocol. Chloroquine was the most effective inhibitor, followed by leupeptin, propylamine, E-64, and pepstatin. When lysosomes were incubated with various combinations of inhibitors, including a weak base and an enzyme inhibitor, a somewhat higher inhibition (86%) was obtained. To assess if lysosomes are active in the degradation of both short and long lived proteins, lysosomes were isolated from livers of rats labeled with [14C]leucine for various time intervals. The highest fractional proteolytic rates were seen for short lived proteins. If the recovery of the isolated lysosomes is taken into consideration, about 80% (short labeling time) and 90% (long labeling time) of the total proteolysis in the homogenate could be accounted for by lysosomes. Isolated Golgi, mitochondrial, and microsomal fractions displayed negligible proteolytic activities. The cytosol contributed one-fifth of the total protein breakdown of short lived proteins, whereas insignificant proteolysis was recovered in the cytosolic fraction following long time labeling. Accordingly, we propose that 1) lysosomal inhibitors do not completely suppress proteolysis in isolated lysosomes and that 2) both short and long lived proteins are degraded in lysosomes.     

Regulation of Intracellular Proteolysis in Escherichia coli

Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli. Generally, NH(4) (+) is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.     

Conformation of poly(L-lysine) homologs in solution

The effect of the number of methylene groups in the side chains on the conformation of polypeptides is assessed for three poly(L -lysine) homologs with R = –(CH₂)_nNH₂. Circular dichroism studies show a pH-induced helix–coil transition in 0.05 M KCl with midpoints at 9.6, 9.0, and 8.7 for n = 5, 6, and 7, respectively, as compared with 10.1 for (Lys)_x (n = 4). Homologs with n = 6 and 7 could be partially helical even when the side groups are fully charged (with n = 7, the compound is highly aggregated above pH 9.1). Thus, the longer the number of methylene groups the more stable is the helical conformation of these homologs. Potentiometric titration of the n = 5 homolog gives a ΔG° of ?310 cal/mol (residue) for the uncharged coil-to-helix transition at 25°C. The corresponding ΔH° and ΔS° are ?1740 cal/mol (residue) and ?4.8 e.u./mol (residue). Unlike (Lys)_x, the uncharged helix-to-β transition is slow and incomplete even after heating at 80°C for 1 hr. Addition of methanol enhances the helical formation in neutral solution with midpoints at 72, 52, and 27% methanol (v/v) for n = 5, 6, and 7, respectively [cf. 88% for (Lys)_x]. Addition of sodium dodecyl sulfate induces a coil-to-helix transition for all three homologs in contrast with the β form of (Lys)_x under similar conditions.     

The breakdown of myelin-bound proteins by intra- and extracellular proteases

Changes in protein components of purified myelin were measured following incubation in vitro with purified intra- and extracellular enzymes. Incubation with calf brain cathepsin D did not result in a significant relese of acid-soluble peptides as measured by ninhydrin analysis but was accompanied by a large loss of myelin proteins as determined on SDS-acrylamide gels. After 5 hr at 37°C there was a loss of about 25% for fast and slow basic proteins and the Agrawal proteolipid, but only a 5–10% loss for the Folch-Lees and Wolfgram components. Rat brain cathepsin D prepared by affinity chromatography gave a 30–60% breakdown of basic proteins and proteolipids. In general, breakdown using lyophilized myelin was increased over two-fold as compared to experiments with fresh myelin. Breakdown induced by cathepsin D was completely inhibited by the pentapeptide pepstatin. Incubation of myelin at physiological pH resulted in an endogenous breakdown of about 12% for basic proteins in freshly prepared, and 50% for lyophilized material. Addition of a soluble neutral proteinase that splits hemoglobin did not induce additional breakdown except for a small change in the Folch-Lees component. The extracellular enzymes pepsin and TPCK-treated trypsin resulted in a larger breakdown of all components as compared to brain enzymes. Present results demonstrate that all protein components of myelin are accessible to hydrolases and vulnerable to breakdown to varying extents by brain enzymes. These facts are consistent with the known rates for myelin protein turnover and may have a bearing on changes associated with demyelinating diseases     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

Autolysis in the tail muscles of metamorphosing tadpoles

Degradation of muscle homogenate from the metamorphosing tadpole tail of bullfrog, Rana catesbeiana, was examined at acid and neutral pHs. More rapid and complete degradation was observed at acid pH. Proteinases working at acid pH were not inhibited by pepstatin but were inhibited by leupeptin. However, the inhibition by leupeptin was enhanced by pepstatin. These results show that lysosomal proteinases, a thiol proteinase(s) rather than cathepsin D, are involved in the degradation of tail muscle proteins.     

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

Abstract

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of e-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally- induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO₄)₂. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from β, to 50% α, to random coil at the maximum methylation.

Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)_n·(dAdT)_n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)_n·(dC)_n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative ψ-spectra are induced, which, in the case of (dGdC)_n·(dGdC)_n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration.

It is suggested that the biological effects of methylated proteins may be evoked by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

Enhanced anti-tumor effects of HPV16E749–57-based vaccine by combined immunization with poly(I:C) and oxygen-regulated protein 150

Background: It is well known that both heat shock protein (HSP) and Toll-like receptor (TLR)3 agonist polyinosinic:polycytidylic acid (poly(I:C)) are capable of promoting the antigen-specific immune responses. In the current study, we assessed whether the anti-tumor effects of the HPV16E7_49–57-based vaccine can be elevated by combined applications of poly(I:C) and oxygen-regulated protein 150 (ORP150) in a mouse cervical cancer model. Methods: Recombinant mouse ORP150 and HPV E7_49–57 peptide were combined to passively form the ORP150–E7_49–57 complex under heat shock conditions. The effects of ORP150–E7_49–57 complex plus poly(I:C) adjuvant on lymphocyte proliferation and functional cytotoxic T cells were investigated by methyl thiazolyl tetrazolium (MTT), ELISPOT, and non-radioactive cytotoxicity assays. Finally, the complex's therapeutic anti-tumor effects with and without adjuvant therapy were observed in a tumor challenge experiment. Results: This combination vaccine approach significantly enhanced the proliferation of splenocytes and induced strong E7_49–57-speci?c CTL responses. More importantly, the ORP150–E7_49–57 complex plus poly(I:C) vaccine format demonstrated more potent anti-tumor effects than ORP150–E7_49–57 complex alone or E7_49–57 plus poly(I:C) in TC-1 tumor-bearing mice. Conclusion: Both poly(I:C) and ORP150 chaperone can synergistically enhance the anti-tumor effects of the HPV16E7_49–57-based vaccine in vitro and in vivo. This strategy provides a platform for the design of a tumor therapeutic vaccine capable of inducing an effective anti-tumor immune response.     

Specific inhibition by NH4Cl of autophagy-associated proteolysis in cultured fibroblasts

Rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH₄Cl inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH₄Cl markedly increased the number of these intracellular vacuoles. Upon release from NH₄Cl inhibition, these cells showed a compensatory enhanced release of ¹⁴C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH₄Cl inhibits the final hydrolysis and release steps in this mechanism.     

The effect of insulinomimetic agents on protein degradation in H35 hepatoma cells

A wide variety of agents are shown to mimic insulin action and inhibit rates of intracellular protein degradation in H35 hepatoma cells. For oxidizing agents such as NaNO₂, H₂O₂ and oxidized glutathione, inhibition of protein breakdown is reversed by adding catalase. Phenylhydrazine behaves like an oxidant and mimics insulin action in a manner potentiated by superoxide dismutase and reversed by catalase. Similarly the effect of insulin itself is increased by superoxide dismutase and reduced by catalase. Sulfhydryl reagents also mimic insulin action: inhibition of protein breakdown is seen following addition of 2-mercaptoethanol or a brief pre-treatment with N-ethylmaleimide or iodoacetate. Mild pre-treatment with trypsin also inhibits subsequent rates of protein breakdown. A model is proposed suggesting that these insulinomimetic actions involve a common mechanism which links the generation of active oxygen species through the redox potential of the cell to the activation of a proteinase.     

Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.     

A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp_(43-58) (penetratin), Tat_(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.