Receptor-mediated endocytosis of enterokinase by rat liver: Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30–45 kDa range.     

3-hydroxy-3-methylglutaryl coenzyme A reductase in human liver microsomes: active and inactive forms and cross-reactivity with antibody against rat liver enzyme

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme catalyzing the rate-limiting step in cholesterol biosynthesis, exists in one active (dephosphorylated) and one inactive (phosphorylated) form in liver microsomes obtained from several animal species. The present study was undertaken in order to determine a) whether the human enzyme also exists in active and inactive readily interconvertible forms; b) whether the large inter-individual variation in HMG-CoA reductase activity observed in normal man can be explained by variations in the activation state of the enzyme; and c) to characterize the reactivity of antibodies raised against rat liver HMG-CoA reductase with the intact human microsomal enzyme. HMG-CoA reductase activity, assayed in microsomes prepared in the presence of 50 mM NaF, was only 17 +/- 3% of the activity observed in microsomes prepared from the same liver in the absence of fluoride. Preincubation of microsomes prepared in NaF with alkaline phosphatase resulted in a tenfold increase of enzyme activity, while the activity of microsomes prepared without fluoride was increased also (by about 45%) with this treatment. On the other hand, the activated enzyme could be inactivated by incubation of microsomes with Mg-ATP. In eleven normal weight, normolipidemic gallstone patients, the HMG-CoA reductase activity determined in microsomes prepared without NaF ("standard procedure") reflected well both the "expressed" activity (in microsomes prepared with NaF) and the "total" (fully activated) enzyme activity; correlation coefficients were +0.80 and +0.84, respectively. Preincubation of human liver microsomes with rabbit antiserum against partially purified HMG-CoA reductase from rat liver resulted in a 72 +/- 6% inhibition of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reorganization of the endocytic compartment in regenerating liver.

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocyte's cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

Preferential localization of rat liver D-myo-inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase in bile-canalicular plasma membrane and ''late'' endosomal vesicles.

Previous studies have shown that most of the inositol 1,4,5-trisphosphate/inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity of rat hepatocytes is associated with the plasma membrane [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. We now show that the specific activity of this enzyme is highest in the bile-canalicular domain of the plasma membrane, at the opposite pole of the hepatocyte from the presumed site of receptor-mediated formation of inositol 1,4,5-trisphosphate. In intact hepatocytes and in sealed membrane vesicles originating from the bile-canalicular domain of the plasma membrane, the 5-phosphatase activity was mostly latent and therefore located at the cytoplasmic surface. A substantial amount of 5-phosphatase was also found in rat liver endosomal fractions, particularly a 'late' endosomal subfraction, indicating that this enzyme may be transported between the sinusoidal plasma membrane and other cellular membranes.     

Effect of ethanol on the DNA-polymerase activity in the liver subcellular fractions of adult and old rats

The effect of 5% ethanol on DNA polymerase activity in nuclei, mitochondria, microsomes and cytosol of intact and regenerating liver of adult and old rats has been studied. No changes in DNA polymerase activity were detected in subcellular fractions of adult rat liver. On the contrary, the increased activity of intact liver nuclei and decreased activity of regenerating liver microsomes was observed with ageing. These age-dependent peculiarities of DNA polymerase activity in response to 5% ethanol may be related to changes in the enzyme molecules or microenvironment associated with ageing.     

Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase.

We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.     

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.     

Modulation of lipid metabolism at rat hepatic subcellular sites by female sex hormones

The present studies examine the modulation of lipoprotein metabolism at subcellular sites in the liver by female sex hormones. Subcutaneous injection of ethinyl estradiol (5 mg/kg) decreased both triacylglycerol (TG) lipase activity and neutral cholesteryl ester (CE) hydrolase activity in hepatic endosomes while increasing lysosomal lipid hydrolysis. These data suggest that estrogen may induce a shift in the site of intracellular lipid catabolism similar to that found in fasting animals [1]. This work also shows that TG-lipase activity is increased in the CURL and MVB endosomal fractions of ovariectomized rats compared to that found in the equivalent endosomal compartments of age-matched intact female controls. These observations are consistent with an inhibition of endosomal lipase by female sex hormones under physiologic conditions.     

ATPase activity of the smooth and rough microsomal subfractions of the heart and liver in rabbits with a short-term microcirculatory disorder]

Short-time disturbances of the microcirculation in rabbits had a different influence on the ATP-ase activity of smooth and rough microsomes of the liver and heart. Mg+2ATP-ase activity in the liver decreased in both microsome fractions; as in the heart, the enzyme activity increased only in the smooth (light) microsomes.     

Calciferol 25-hydroxylase activity in smooth and rough endoplasmic reticulum of rat liver.

Calciferol 25-hydroxylase activity of vitamin D-deficient rats was measured in both liver microsomes and submicrosomal fractions. The smooth and rough-surfaced microsomes were prepared by a density gradient centrifugation technique in the presence of cesium chloride. Purity of the isolated microsomal membranes was ascertained by electron microscopy, RNA determination, measurement of enzyme markers, and by labeling of the cytoplasmic RNA with [5-³H]orotic acid. Calciferol 25-hydroxylase activity was present in both smooth and rough-surfaced microsomes. The specific activity of the enzyme was greater in the rough fraction. There was a linear relation between enzymic activity and the concentration of enzyme for both total and submicrosomal fractions. These data show the presence of calciferol 25-hydroxylase activity in both smooth and rough-surfaced microsomes isolated from livers of vitamin D-deficient rats.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.     

Major and Minor Receptor Group Human Rhinoviruses Penetrate from Endosomes by Different Mechanisms

Intercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111–123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS : II. The Site of Phospholipid Synthesis in the Initial Phase of Membrane Proliferation

The specific activity of the acyltransferases of smooth microsomes of rat liver rose threefold by 12 h after injection of phenobarbital, while the activity of the acyltransferases of the rough microsomes rose slightly to peak at 3–4 h, and subsequently fell. The latter rise was abolished by treatment of the animal with actinomycin D or puromycin, while that of the smooth microsomes was unaffected. Incorporation of [¹⁴C]glycerol into phospholipid of smooth microsomes was elevated 100% by phenobarbital, while that of the rough microsomes was elevated 15%, and this could be accounted for by exchange between the microsomal phospholipids. The phospholipid/protein ratio of the smooth microsomes rose 1.5 times 3–4 h after injection of phenobarbital, while that of the rough microsomes fell slightly. The specific activity of NADPH cytochrome c reductase and NADPH diaphorase rose first in the rough microsomes, and subsequently in the smooth microsomes at a time coinciding with the return of the phospholipid/protein ratio to the control level. The rise in phospholipid/protein ratio was unaffected by actinomycin D or puromycin. These results indicate that the proliferating smooth membranes are the site of phospholipid synthesis, and that the phospholipid/protein ratio of these membranes may change independently.     

Different developmental changes in latency for two functions of a single membrane bound enzyme: glucose-6-phosphatase activities as a function of age.

(1). The capacity for the synthesis of glucose 6-phosphate from PPi and glucose as well as for glucose-6-P hydrolysis, catalyzed by rat liver microsomal glucose-6-phosphatase, increases rapidly from low prenatal levels to a maximum between the second and fifth day, then slowly decreases to reach adult levels. When measured in enzyme preparations optimally activated by hydroxyl ions, the maximum neonatal activities were 4--5-fold higher than in adult animals and several-fold higher than had previously been observed for the unactivated enzyme. (2) The latencies of two catalytic activities associated with the same membrane-bound enzyme show strikingly different age-related changes. The latency of PPi-glucose phosphotransferase activity reaches high levels (60--80% latent) soon after birth and remains high throughout life, while the latency of glucose-6-P phosphohydrolase decreases with age. The phosphohydrolase is 2--3 times more latent in the liver of the neonatal animal than in the adult. (3). The well established neonatal overshoot of liver glucose-6-phosphatase is almost entirely due to changes in the enzyme in the rough microsomal membranes. The enzyme activity in the rough membrane reaches a maximum and then decreases after day 2, while that in the smooth membrane is still slowly increasing. Despite the great differences in absolute specific activities and in the pattern of early enzyme development between the rough and smooth microsomes, enzyme latency in the two subfractions remains parallel, glucose-6-P phosphohydrolase being only slightly more latent, while PPi-glucose phospho-transferase is much more latent in smooth than in rough membranes throughout life. (4). Kidney glucose-6-P phosphohydrolase and PPi-glucose phosphotransferase activities were found to change in a parallel fashion with age, showing a small neonatal peak between days 2 and 7 before rising to adult levels. Kidney phosphotransferase activity, like that of liver, remained highly latent throughout life. In contrast to liver, the glucose-6-P phosphohydrolase of kidney did not show a characteristic decrease in latency with age and in the adult remained appreciably more latent than in liver. (5). An improved method was devised for the separation of smooth microsomes from liver homogenates.     

Demonstration that the insulin receptor undergoes an early structural modification following insulin binding

Processing of the insulin receptor by hepatocytes was studied using a 125I-labelled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350 000 molecular weight) and binding (alpha) subunit (135 000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140 000 (non-reduced) which on reduction generated species of 100 000 and 68 000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.     

Evidence that the enzymes involved in the methylation of phosphatidylethanolamine are on the external side of the microsomal vesicles

The topology of the phosphatidylethanolamine (PE)-N-methyltransferase(s) on rat liver microsomes has been studied. The activity for the conversion of PE to phosphatidylcholine decreased by 50% after 2 min of exposure of the microsomes to trypsin and was virtually eliminated with 15 min. When exogenous monomethyl-PE or dimethyl-PE were incubated with microsomes, the formation of dimethyl-PE and phosphatidylcholine were also eliminated as a result of trypsin digestion. During the experiments the microsomes remained intact, since the latency of the mannose-6-phosphate phosphohydrolase remained approx. 90%. It is concluded that the active site(s) of the enzyme(s), or portions of the enzyme(s) indispensable to its activity, are present at the cytosolic side of the microsomes.