Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Time course studies revealed that at 30 s after intraportal injection of 200 μU of ¹²⁵I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700g (nuclei + debris), 10,000g (mitochondria + lysosome), 105,000g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10^?3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after ¹²⁵I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after ¹²⁵I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of ¹²⁵I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 mm reduced glutathione and 15 mm EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected ¹²⁵I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.     

ATP-induced lysis of rat parotid secretory granules: Possible role of ATP in exocytotic release

Secretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP-induced lysis, has been studied most extensively using adrenal chromaffin-granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP-induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media ATP-induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin-granule process:

• 1 It is a saturable function of ATP with half-maximal rates observed at 0.5 ± 0.1 mM ATP.
• 2 It is temperature dependent, eg, r = 6.1 ± 2.1%/min at 30°C vs 12.2 ± 2.5%/min at 37°C.
• 3 It is inhibited in hyperosmotic media, eg, r = 5.3 ± 0.3%/min at 0.3 OsM vs 0.8 ± 0.2%/min at 0.4 OsM.
• 4 It shows a nucleotide preference of ATP = GTP > ADP > AMP > CTP = ITP.
• 5 It has an anion requirement.

The above findings, combined with reports of ATP-induced lysis of cholinergeric, insulin, and posterior-pituitary vesicles, imply that ATP-induced lysis may reflect an ATP-dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent to which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP-induced lysis in this and other secretory-vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.     

Biosynthesis of biopterin in Ascaris lumbricoides suum

In in vivo experiments, radioactivity from [U-¹⁴C]GTP was incorporated into biopterin, and, in fact, all carbon atoms of biopterin synthesized in Ascaris lu lumbricoides suum originated from GTP.Biopterin was also biosynthesized in homogenates of tissue fluid and muscles of Ascaris lumbricoides suum.The enzyme which catalyzes sepiapterin synthesis from D-erythto-7,8-dihydroneopterin-3′-phosphate was found in A. lumbricoides suum extracts and extracted in the 0–30% (NH₄)₂SO₄ fraction from a 40 000 × g supernatant. The enzyme was purified by Sephadex G-200 column and DEAE-cellulose column chromatography. It is suggested that sepiapterin could be an intermediate compound in biopterin biosynthesis.     

Post-secretory acquisition of apolipoprotein E by nascent rat hepatic very-low-density lipoproteins in the absence of cholesteryl ester transfer

Previous work has shown that nascent hepatic very-low-density lipoproteins (VLDL) in the rat are biosynthesized without the obligatory co-factor (apolipoprotein C-II) for lipoprotein lipase-mediated hydrolysis of their core triacylglycerols. Upon secretion, apolipoproteins C-II and C-III are rapidly transferred to the particles from high-density lipoprotein (HDL) within the space of Disse and upon the entry into the plasma. Here we extend those studies to include observations on the apolipoprotein E content and lipid composition of nascent hepatic VLDL before and after exposure to plasma components. We have elected to use hepatic secretory vesicle VLDL rather than liver perfusate VLDL as truly representative of the nascent lipoproteins. Nascent VLDL from fed rats has an apolipoprotein B/E ratio of 6.6 ± 0.5, whereas that from fasted animals is 13.9 ± 2.3. Incubation of nascent VLDL from fed and fasted rats with d > 1.063 g/ml rat serum, HDL or the d > 1.21 g/ml fraction resulted in a mass transfer of apoliproprotein E to the VLDL such that the apolipoprotein B/E ratio decreased to at least that of serum VLDL (3.4 ± 0.3). The d > 1.21 g/ml fraction appeared to contain a species of apolipoprotein E which most actively transferred to VLDL. The acquisition of apolipoprotein E by nascent secretory vesicle VLDL was attended by a loss of phospholipids, particularly the C₄₀ (stearoylarachidonyl) molecular species, and an increase in the cholesterol-to-phospholipid ratio from 0.11 ± 0.01 to 0.18 ± 0.03. No evidence was obtained to suggest a simultaneous acquisition of cholesteryl esters upon incubation of nascent VLDL with VLDL-free serum. We conclude that nascent hepatic VLDL is modified after secretion by acquisition of apolipoproteins C-II, C-III and E with a concomitant loss of phospholipids.     

Some differences in intracellular distribution and properties of the chymotrypsin-like esterase activity of human and rabbit neutrophils

The intracellular localization and properties of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase acitivity) of the rabbit peritoneal neutrophil has been studied and shown to differ from that of the human neutrophil.The major portion of the esterase activity in the rabbit neutrophil is in the 100 000 × g supernatant fraction with distinctly less activity in the lysosomal fraction. The 100 000 × g supernatant contained the highest relative specific activity of any of the subcellular fractions. Rabbit peripheral blood neutrophils gave the same distribution.The 100 000 × g supernatant esterase is 95% esterase 1 and 5% esterase 3, whereas, the lysosomal esterase is 78% esterase 1, 10–16% esterase 2 and 9% esterase 3 as defined by their ability to be inhibited by p-nitrophenyllethyl-5-chloropentylphosphonate. The 100 000 × g supernatant The 100 000 × g supernatant and lysosomal esterase activities further differ in their susceptibility to other inhibitors, their pH optima, ease of elution from DEAE and isoelectric points. Two molecular weight species of 174 000 and 70 000 were found in the 100 000 × g supernatant fraction and extracts of the lysosomal fraction but usually in differing proportions.In confirmation of others, essentially all of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase activity) of the human neutrophil is in the lysosomal fraction, unlike the rabbit cell. The human neutrophil esterase was less susceptible to inhibition by p-nitrophenylethyl-5-chloropentylphosphonate and diisopropylphosphofluoridate but more susceptible to soybean trypsin inhibitor than rabbit esterase activity. The pH optimum of the human neutrophil esterase differed from either the rabbit lysosomal or 100 000 × g supernatant esterase, as did the isoelectric point and molecular weights.     

Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.     

The isolation of intact adrenal chromaffin granules using isotonic Percoll density gradients

An improved method for the isolation of intact adrenal chromaffin granules under isotonic conditions, using a Percoll density gradient, is presented. After dissection, homogenization, and differential centrifugation, the crude granule homogenate was layered onto a gradient medium previously centrifuged at 20,200g × 5 min, consisting of 30% ($\text{v}\text{v}$) Percoll, 0.27 m sucrose, and 10 mm Tris-maleate, pH 7.0. After centrifugation for 40 min at 8650g in a standard preparative centrifuge, the chromaffin granules were found to band in the lowest fraction, where 45% of the catecholamines and 60% of the ATP could be recovered. With respect to other published methods, the percentage of lysosomal and mitochondrial contamination compared favorably. In addition, granules isolated by the Percoll gradient method were found to have at least 42 and 14% higher ATP and catecholamines, respectively, per milligram of protein. It is suggested that this method offers the advantages of ease of preparation, purity, and cost efficiency when compared with previously published techniques.     

Calpain activity alters in rat myocardial subfractions after ischemia or reperfusion

To examine whether calpain is activated during ischemic or reperfusion injury, we measured calpain activity of the subfractions of rat myocardia after global ischemia for 60 min or the ischemia followed by 30 min reperfusion by the Langendorff procedure. The myocardial homogenate was fractionated into 600 × g, 10 000 × g and 100 000 × g pellet fractions as well as 10 000 × g supernatant fraction. The supernatant fraction was further subjected to DEAE cellulose and phenyl-Sepharose chromatographies to separate μ- and m-calpains. The m-calpain activity of the DEAE fractions after global ischemia for 60 min was higher but that after ischemia-reperfusion was lower than that of the control. On the other hand, the ischemia-reperfusion but not ischemia by itself raised the calpain activity of the phenyl-Sepharose fraction (μ-calpain) and the 10 000 × g pellet measured at 100 μM and 5 mM Ca²⁺. Treatment with verapamil but not with ryanodine during ischemia attenuated the increase in m-calpain activity. A dot-blotting analysis of calpain antigenicity showed a decrease in soluble but no change in the particulate fractions after ischemia-reperfusion. An immunoblotting technique did not detect proteolysis of the calpain 80-kDa subunit. These observations suggest that calpain is activated by Ca²⁺ influx during ischemia and reperfusion without gross changes in its amount. Some unknown processes other than translocation or autolysis are thought to be involved in the alterations.     

Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73–80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5–2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO₃ ^? > Br^? > I^? > Cl^? and was facilitated by acidic pH. The anion selectivity for NO₃ ^? and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid secretory granules.     

Properties of membrane phospholipids and their fatty acyl compositions of secretory granules from rat parotid gland

A secretory granular fraction isolated from rat parotid glands was remarkably different from a microsomal fraction in its phospholipid composition. It had higher levels of lysophospholipids (8%) and phosphatidylethanolamine (31%), while there were lower levels of phosphatidylcholine (40%) and phosphatidylserine (2.1%) than the microsomal fraction. However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be nearly similar to each other. ESR analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. The relevance of these observations to physiological function of secretory granules is discussed.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

Isolation of Cytoplasmic Pituitary Granules with Gonadotropic Activity

A fraction isolated from the anterior pituitary glands of rats castrate for 8 weeks contained essentially a single cytoplasmic constituent with which the major portion of the gonadotropic hormone activity was associated. The glands were homogenized in an 0.25 M sucrose + 7.3 per cent polyvinylpyrrolidone (PVP) solution and fractionated by differential centrifugation to give a heterogeneous small granule fraction which contained almost all the gonadotropic hormone activity. The active supernatant containing this small granule fraction was separated into layers by isopycnic gradient centrifugation on a continuous 6 to 45 per cent sucrose + 17.5 per cent "diodrast" + 5 x 10^-4 M "versene" gradient at 100,000 g for 2 hours. Three layers were obtained and the pellet from the active bottom layer was sectioned, examined with the electron microscope, and found to contain 200 mµ granules, mitochondria, ergastoplasm, and other cellular debris. This layer was fractionated further by isopycnic and differential centrifugation to obtain a pellet which contained the major portion of the gonadotropic hormone activity. Because of the heterogeneity of this fraction, due to the contamination of the 200 mµ granules with mitochondria and other cellular debris, the active layer and the resuspended active pellet, obtained by centrifuging this layer first at 17,000 g then diluting the supernatant and centrifuging at 30,000 g for 1 hour, were filtered through Millipore HA paper with a pore size of 0.45 µ. The cytoplasmic material containing the gonadotropic hormone activity passed through the filter paper and this activity was recovered in the pellets obtained by centrifuging at 100,000 g for 1 hour. These active pellets consisted almost entirely of 200 mµ granules with a minimum amount of contamination, and they contained the major portion of the gonadotropic hormone activity with practically none remaining in the supernatant fraction. These results are discussed in view of their importance to the cytology of the pituitary gland.     

A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris

This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut^s Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25 % of the total proteins in the culture supernatant, after 120 h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45 % of the total culture supernatant proteins) with concomitant reduction in the induction time to 48 h. The amount of BbST obtained was 148 mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Mut^s strains of P. pastoris.     

Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80–90% mitochondria, 5–10% platelets, and 5–10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.     

Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis

Summary Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B,N-acetyl-β-glucosaminidase, and β-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis. In control rats high enzymatic activities were found in the postnuclear 1000g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis was found.     

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Distribution and nonphotochemical transformation of phytochrome in subcellular fractions from pisum epicotyls

In etiolated pea (Pisum sativum L. cv. Alaska) shoots about 3% of the total extractable phytochrome was found in the mitochondrial fraction and about 4.5% in the microsomal fraction, while over 70% was soluble in the 105,000g supernatant. The value of Δ(ΔA) per milligram of protein was significantly higher in the 105,000g supernatant than in these particulate fractions. The percentage conversion of Pr to Pfr was approximately proportional to the total dose of red light in every subcellular fraction tested, unless the dose approached a saturation level. After a brief irradiation of intact shoots with red light at 26 C, each subcellular fraction showed different patterns of dark transformation in vivo at 26 C; that is, the amount of the particulate-bound phytochrome increased immediately after the irradiation, and a reversion of Pfr to Pr was indicated for the first 2 hr in the 12,000g supernatant, but not at all in the mitochondrial and microsomal fractions. The amounts of Pr in the mitochondrial and microsomal fractions did not change during the dark incubation, while those in the 12,000g supernatant increased with time. Similar results were obtained with apical shoot segments after exposure to red light at 0 C and a subsequent dark incubation on moist filter paper at 26 C.