Insulin-degrading neutral protease from plasma membranes of rat liver cells and erythrocytes

Insulin-degrading neutral proteinase with molecular weight of 70 kDa was partly purified from the rat liver and erythrocyte plasma membranes. Incubation of membranes with [gamma-32P]ATP resulted in the enzyme phosphorylation. Intensity of this process greatly increased in the presence of insulin (100 microU/ml), and correlated with the elevation of the insulin-degrading activity in proteinase. Ca2+, Mn2+, dithiothreitol, cysteine were shown to have a stimulatory effect on insulin degradation; p-chloromercuribenzoate significantly repressed this process. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor did not affect the activity of the proteinase. It was concluded that the investigated enzyme was a calpain and may participate in the mechanism of insulin action.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Influence low-intensity laser irradiation on the ultrastructural organization of loach embryo cells

The ultrastructural organization of loach embryo cells (Misgurnus fossilis L.) at the stage of the first and the tenth embryo divisions was investigated under the influence of low intensity helium-neon laser irradiation of 5 min exposure. It was determined that the effect of laser irradiation led to ultrastructural changes: the shape of mitochondria and multivesicular bodies changes. The obtained results explain the possible mechanism of influence of low-intensity laser irradiation at the cellular level.     

Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver cells.

A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5'-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane.     

Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in Vmax (i.e., maximal enzyme activity) without a change in Km (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.     

Phosphorylation of lactate dehydrogenase by purified plasma membranes of loach liver cells: stimulation with insulin

It is established that insulin enhances the ability of the loach liver plasma membranes to phosphorylate lactate dehydrogenase. In the case of insulin-treated plasma membranes the amount of incorporated 32P is more than 4 times higher than that of the basal level. It is concluded that insulin-stimulated plasma membrane-dependent phosphorylation of the enzyme is one of the possible molecular mechanisms of hormone action on intracellular metabolism.     

Localization of neutral,magnesium-stimulated sphingomyelinase in plasma membrane of cultured neuroblastoma cells

A parallel is shown between the distribution of neutral sphingomyelinase and plasma membrane enzymes (5′-nucleotidase and (Na⁺ + K⁺)-activated ATPase) in cultured neuroblastoma cells. In contrast there is no evidence of localization in lysosomes (β-hexosaminidase and acid sphingomyelinase), mitochondria (carnitine palmitoyltransferase), or cytosol. Activity in the microsomal fraction is attributed primarily to plasma membrane contamination.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.     

Evidence that neutral spingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg²⁺-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, α-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg²⁺-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases.

Proteinase contaminants in some plasma albumin samples have previously been shown to produce cleavage of the albumin molecule at acid pH. The F conformer, existing at pH 3.8, is cleaved near erisidue number 400 to yield a large N-terminal fragment of approximately 46,000 daltons. No cleavage was found at pH above approximately 4.4. It is shown in this paper that the proteinase contaminants are active over a broad pH range from 2.5 to 11.4 provided conditions are such as to induce some breakdown of the native conformation of the albumin molecule. Addition of Tris-borate buffer (0.1 M) at pH 7.5-9 is sufficient to permit cleavage. At pH near 9 this occurs predominantly 42,000 and 27,000 daltons. Near neutral pH substantial cleavage occurs in 4-8 M urea solution or in the presence of sodium dodecyl sulfate (AD110 complex). Under these conditions there are two large fragments (42,000 and 47,000 daltons) and essentially two small ones (20,000-27,000 daltons). Under conditions where there is no cleavage at 38-40 degrees, substantial cleavage results at 50-65 degrees but enzyme inactivation also occurs toward the top of this range. The alkaline activity is inhibited by soybean trypsin inhibitor but not by pepstatin; the reverse is true of the low pH activity. Cleavage at neutral or alkaline pH under the various conditions occurs primarily at X-Leu bonds while the low pH activity was already shown to occur at X-Phe. These facts suggest the presence of at least two enzymes. There is surprisingly little pH dependence over the range 7.5-9 in any of the media examined, even though albumin is known to undergo a significant conformational change in this range, the N leads to B transition. This transition is thought to be essentially a tertiary change with little loss of helix content. It is suggested that loss of native secondary structure, especially uncoiling of helical regions, is crucial to permit attack by these enzymes.     

Sigma receptors of the loach embryo regulate ornithine decarboxylase activity

When loach embryos (Miagurnus fossilis) were incubated with opioid substance, sigma-receptors ligand SKF 10.047, activity of the key enzyme of polyamine synthesis - ornithine decarboxylase (ODC), increased. A correlation between teratogenic and ODC-stimulated action of SKF 10.047 was stage-specific and depend on opioid concentration. Possible mechanism of embryonic cell aggregation under SKF 10.047 action and polyamines role in that process is discussed.     

Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.