Insulin degradation in insulinoma: evidence for the occurrence of an inactive form of glutathione-insulin transhydrogenase and for the absence of insulin A and B chains degrading protease(s)

Islet cell tumors (insulinomas) have been found to contain insulin-degrading activity. Apparent K_m values for insulin obtained with tumor extracts were similar to those found for other tissues and for purified glutatione-insulin transhydrogenase (GIT). In Ouchterlony double diffusion experiments with antibody to purified human liver GIT, each tumor extract gave a single precipitation band of identity with purified human liver GIT. Examination by chromatography on Sephadex G-75 of the products formed from ¹²⁵I-insulin upon incubation with tumor extracts showed the same products (A chain, and B chain rich-A chain aggregate) as previously found with purified GIT; however, there was no further degradation (i.e., proteolysis) of A chain to low molecular weight components. These results indicate that the insulin-degrading activity present in the islet tumor is, in fact, GIT and that the protease(s) that further catabolizes the insulin A and B chains is apparently missing in the insulinoma. These data could be interpreted to indicate that the function of GIT in this tissue is to promote the biosynthesis of proinsulin and insulin rather than their degradation. Data are also presented which indicate that in insulinoma GIT is present in an inactive state as a divalent metal ion complex since it could be activated with EDTA and/or GSH.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor.

Membrane preparations containing essentially only the four polypeptides considered to constitute the acetylcholine receptor are purified from $\text{Torpedo}\text{californica}$ electroplax. Treatment of these membranes with 2% ($\text{w}\text{v}$ aqueous sodium cholate followed by removal of all insoluble matter results in a solubilized purified receptor preparation that can be reassociated with phospholipids during dialysis to remove the detergent. Such reconstituted receptor is shown to retain the capability of translocating ²²Na⁺ across the membrane in response to carbamylcholine binding in a highly reproducible manner. The dose response for this effect is similar to that observed for the original electroplax membrane preparation and the carbamylcholine induced signal is completely blocked by α-bungarotoxin.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

In vivo chain elongation of hepatic DNA: 1-beta-D-arabinofuranosyl cytosine sensitive steps.

The $\text{in vivo}$ chain elongation of rat liver DNA following partial hepatectomy was studied using alkaline sucrose gradients. DNA made in 5 min was less than 4 × 10⁷ daltons and that made in 30 min was heterodisperse and by 4 hr 75% of the DNA became larger than 1 × 10⁹ daltons. Administration of 1-β-D-arabinofuranosyl cytosine (ara-C) 5 min after thymidine-³H injection inhibited the chain elongation, whereas if given 30 minutes after thymidine-³H pulse did not inhibit the chain elongation. Thus the $\text{in vivo}$ chain elongation of rat liver DNA consists of at least two steps 1) a step sensitive to ara-C involving nucleotides addition and 2) the other insensitive to ara-C and probably involving ligation of polynucleotide chains.     

Glutathione peroxidase activity of glutathione-S-transferases purified from rat liver

Gel filtration chromatography demonstrated the presence of two peaks of glutathione peroxidase activity assayed with cumene hydroperoxide in the soluble fraction of rat liver, brain, kidney, and testis. The peak with an approximate molecular weight of 45,000 (GSH-Px II) was purified from rat liver labeled $\text{in vivo}$ with Na₂⁷⁵SeO₃. Chromatography on DEAE-cellulose, Sephadex G-150, DEAE-cellulose, and CM-cellulose resulted in the co-purification of glutathione-S-transferase activity measured with 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity assayed with cumene hydroperoxide, and in the removal of all detectable ⁷⁵Se. Studies on GSH-Px II indicated that the apparent K_m for both cumene and t-butyl hydroperoxides was considerably higher than that for purified seleno-glutathione peroxidase. The V_max estimated with cumene hydroperoxide was only $\text{1}\text{300}$ of that determined for the selenoenzyme at pH 7.5 and with 1 mM GSH.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.     

Vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase and other ATPases.

Vanadate is a potent inhibitor of the Ca²⁺-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca²⁺ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca²⁺Mg²⁺-ATPases of the ascites and human red cell plasma membranes, Mg²⁺-ATPase of the ascites plasma membrane, and the K⁺-ATPases of $\text{E.}\text{coli}$ and hog gastric mucosal cell membranes. The ascites plasma membrane Ca²⁺-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.     

Assay of catechol O-methyltransferase by determination of the m-and p-O-methylated products using high-performance liquid chromatography.

A new assay of catechol O-methyltransferase (EC 2.1.1.6) is described by determination of the m- and p-O-methylated products of 3,4-dihydroxybenzoic acid. The method involves DEAE-Sephadex A-25 chromatography and reversed-phase high-performance liquid chromatography on a LiChrosorb 5 RP 18 column. The liquid chromatographic solvent system consisted of 0.05 m acetic acid in methanol:water (1:4, $\text{v}\text{v}$), pH 3,2. meta/para ratios of O-methylation are easily obtained by this method. Dimethylation has not been observed with a partially purified enzyme preparation from rat liver.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Plasma membranes from cardiac cells in culture. Enzymatic radio-iodination,evaluation of preparation and properties of the sarcolemma

Plasma membranes from heart (sarcolemma) were prepared by the method of Kidwai, A.M. ((1975) Methods in Enzymology (Fleischer, S. and Packer, L., eds.), Vol. XXXIA, pp. 134–144, Academic Press, New York). On many occasions the sarcolemmal fraction identified by the enzyme markers such as $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ banded at heavier densities ($\text{3 > 1.25}\text{g/ml}$) than expected for plasma membrane ($\text{d < 1.15}\text{g/ml}$). Radio-iodination of the membrane was added as an independent marker and conditions for the reproducible preparation of the sarcolemma were studied. Cultured heart cells were enzymatically iodinated under conditions which did not affect viability and labeled primarily the sarcolemma. The distribution of radioactivity in homogenates of cultured cells on the density gradient corresponded to that of the enzymes' activity. The best sarcolemma preparation was obtained with 0.3 M KCl extraction of heart homogenates in the presence of 0.05 M pyrophosphate, especially if the salt was also present during the fractionation by density gradient centrifugation. Alterations in the density were also observed with erythrocytes and cultured liver cells' plasma membrane. The data suggests a meta-stable state of the plasma membranes due to handling or storage which could cause alterations of some of their physical properties (e.g. density).     

Liver plasma membranes from essential fatty acid-deficient rats: Isolation,fatty acid composition,and activities of 5′-nucleotidase,ATPase and adenylate cyclase

To determine whether changes in unsaturation of fatty acids in rat liver plasma membranes might alter activities of membrane-associated enzymes, liver plasma membranes were prepared from rats fed purified diets lacking or supplemented with essential fatty acids. Two methods of membrane purification were used. A similar degree of purification was obtained with both methods for both depleted and control membranes, as indicated by marker enzyme purification. The proportion of essential fatty acids of the linoleate series was significantly lower in phospholipids from depleted rats. The specific activity of 5′-nucleotidase was lower, and the activity, $\text{V}$ and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for total $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ were higher in the depleted liver plasma membranes. Arrhenius plots of total ATPase activity showed a discontinuity at the same temperature for both the depleted and control membranes. Activity with the depleted membranes was higher at all temperatures tested. Supplementation of deficient rats with a source of essential fatty acids (corn oil) restored $\text{V}$ and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values to normal. Adenylate cyclase activity in the presence of fluoride, glucagon or glucagon plus GTP was significantly lower in the depleted plasma membranes.     

Mechanism of action of cesalin, an antitumor protein.

A protein, cesalin, isolated from $\text{Caesalpinia}$$\text{gilliesii}$ is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na⁺, K⁺-ATPase (ATP phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na⁺, K⁺-ATPase. The Na⁺, K⁺-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5′-Nucleotidase and Mg⁺⁺-ATPase are not inhibited by cesalin in any cells tested.     

Mutagenicity of 3a,8a-dihydrofuro[2,3-b]benzofuran, a model of aflatoxin B1, for Salmonella typhimurium TA100.

Racemic 3a,8a-dihydrofuro[2,3-b]benzofuran has been chemically synthesized as a model of the vinyl ether structure of aflatoxin B₁ (AFB₁) and tested for mutagenicity. In the presence of 9000g rat liver supernatant fraction the compound induced his⁺ revertant colonies in $\text{S. typhimurium}$ TA 100 but with only one five-thousandth the activity of AFB₁. No mutagenicity was found when strain TA98 was used. Omission of the rat liver preparation abolished mutagenic activity. The reduced compound, tetrahydrofurobenzofuran, was inactive as a mutagen either in the presence or absence of the rat liver supernatant.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.