Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Postnatal development of some membrane-bound enzymes of rat liver and kidney

1. The development of rat liver acyl-CoA:sn-glycerol-3-phosphate-O-acyl-transferase (EC 2.3.1.15) is characterized by an increase and decrease in activity during the neonatal period, followed by a second increase and decrease during the late weaning period. Kidney acyltransferase exhibits a similar peak in activity during the neonatal period before increasing to adult levels of activity during the late weaning period. 2. Nucleosidediphosphatase activity increases rapidly during the neonatal period and thereafter gradually rises to adult levels in both liver and kidney. The latency of the enzyme increases rapidly after birth and thereafter shows little change with age. The enzyme appears to be more latent in the liver than in the kidney at all ages studied. 3. NADPH-cytochrome c reductase of liver has a single steep maximum and minimum in activity during the neonatal period, before increasing again to adult levels during the late weaning period. The enzyme in kidney shows a similar developmental pattern but at much lower levels of specific activity. 4. sn-Glycerol-3-phosphate acyltransferase activity was significantly higher in rough than in smooth membranes throughout the neonatal period of rapid smooth membrane proliferation. This distribution of enzyme activity is unlike that reported by others in phenobarbital-induced smooth membrane proliferation and suggests a major role for rough membranes in phospholipid synthesis during the neonatal period. 5. The qualitative similarity in development in rough and smooth microsomal subfractions for each of these enzymes is in distinct contrast with results previously reported for glucose-6-phosphatase.     

Phosphorylation of liver plasma membrane-bound calmodulin

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.     

Apical plasma membrane-bound enzymes of rabbit uterine epithelium

Summary In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, -glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5–8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals.All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the receptive state, 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.Abbreviations d p.c. days post coitum - d. p. hCG days post hCG injection - hCG human chorionic gonadotropin - aP alkaline phosphatase - ATPase adenosine triphosphatase - Ca²⁺-ATPase Ca²⁺-activated adenosine triphosphatase - APM aminopeptidase M - GGT -glutamyl transferase - DPP IV dipeptidyl peptidase IV - PCMB p-chloromercuric benzoate - DFP di-isopropylfluorophosphate - DMF dimethylformamide     

Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.     

Dynamics aspects of long distance functional interactions between membrane-bound enzymes.

The aim of this mini review is to study how an organized charged milieu, such as a membrane, may alter functional long-distance interactions between bound enzymes. Two questions are more specifically considered. The first is to know whether the overall response of a bound enzyme is dependent upon the degree of spatial order of fixed charges and enzymes molecules. The second is to determine whether electric interaction between the fixed charges of the matrix and the charged substrate may generate hysteresis loop of substrate concentration as well as oscillations of this concentration at the surface of the membranes. These effects that have been shown to occur at the surface of membranes, are not the result of intrinsic properties of enzymes. They appear as the consequence of the interplay between functional long-distance interactions between bound enzyme systems and electric repulsion effects of mobile ions. They may be viewed as supramolecular devices that allow storing information from the external milieu.     

A membrane-bound protein inhibitor of the high affinity Ca ATPase in rat liver plasma membranes

The Ca ATPase from rat liver plasma membranes has been recently characterized and partially purified in our laboratory and was shown to depend on a membrane-bound protein activator (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). In the present study, we report that a factor derived from ammonium sulfate washings of rat liver plasma membranes inhibits the partially purified enzyme activity measured in the presence of activator. This factor is a protein as judged by its sensitivity to heat and trypsin. A molecular weight of 29,000 was determined by sucrose gradient centrifugation and gel chromatography. The action of the inhibitor is due to a decrease in the maximal velocity of the enzyme reaction and is reversed by an excess of the activator associated with the enzyme. An important point in the mode of action of this inhibitor is its absolute dependence on magnesium, which most probably explains the difficulty in detecting the plasma membrane Ca ATPase when MgCl2 is added to the assay medium.     

Soluble and membrane-bound neuraminidase in regenerating rat liver]

Two forms of neuraminidase (soluble and membrane-bound) are found in regenerating rat liver. Specific activity of the soluble form was found to be maximal in 18 hours after partial hepatectomy, and that of the membrane-bound form-in 24 hours after the operation. Maximal specific activities of both neurominidase forms from regenerating rat liver considerably exceeded that from intact rat liver, shem-operated liver and also from embryonic and lactating rat liver.     

A novel adenylylation process in liver plasma membrane-bound proteins

Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with [alpha-32P]ATP as well as with [gamma-32P]ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue [alpha, beta-methylene]ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with [alpha-32P]ATP are glycoproteins bound to the cell plasma membrane.     

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes

Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.