Chase Incubation Research Articles

Studies on the biosynthesis of pancreatic glucagon in the pigeon (Columba livia).

The biosynthesis of glucagon was studied in microdissected pigeon pancreatic, islets. [3H]-Tryotophan and [3H]leucine were incorporated into big and little glucagon. No precursor-product relationship was evident between big and little glucagon after radioactive pulsechase and immunoreactive chase incubations. Radioactive and immunoreactive little glucagon and immunoreactive big glucagon were actively secreted and the synthesis of both glucagons was inhibited by high concentrations of glucose. [3H]Tryptophan and [3H]leucine were incorporated into an islet protein of about 20000mol.wt. Gel filtration of extracts of turkey pancreas revealed the presence of an immunoreactive peak of mol.wt. approx. 20000. This glucagon-immunoreactive component was also present in dog and ox pancreas and was stable to chaotropic agents and elution at various pH values. A similar-sized glucagon-immunoreactive species was present in the dog circulation. These results are discussed in the light of the presently accepted mechanisms of glucagon biosynthesis.

Evidence of sequential metabolic cleavage of proglucagon to glucagon in glucagon biosynthesis.

Following a 30 min preincubation in medium containing no isotopes, anglerfish islet tissue was incubated in the presence of [3H]tryptophan and [14C]isoleucine for 20 min. A portion of the tissue was removed for immediate extraction. The remainder was washed thoroughly with unlabeled medium and post-incubated in medium containing an excess of unlabeled tryptophan and isoleucine for varying periods of time. The distribution of radioactive proteins in alcoholic tissue extracts was analyzed by gel filtration and polyacrylamide gel electrophoresis. The distribution of immunoreactive glucagon was determined by radioimmunoassay. Following the 20 min pulse incubation, only proinsulin was labeled with [14C]isoleucine. Two glucagon immunoreactive molecules, one larger than proinsulin (mol wt near 11,400) and the other slightly smaller than proinsulin (mol wt near 9,000), were the primary proteins labeled with [3H]tryptophan following the 20 min. pulse. During chase incubations of increasing duration, 3H-radioactivity appeared in a glucagon immunoreactive molecule with the approximate molecular size of glucagon and increased with chase time while radioactivity in the 11,400 mol wt tryptophan-labeled molecule decreased. With increasing chase time, the 3H-radioactivity attributable to the 9,000 mol wt tryptophan-labeled molecule initially increased and subsequently decreased which is consistent with the pattern that would be expected for a conversion intermediate. The presence of glucagon immunoreactivity in [3H]tryptophan-labeled molecules having molecular weights near that of proinsulin was established by radioimmunoassay of alternate gel slices following electrophoresis of labeled proteins recovered from the proinsulin containing portions of gel filtration eluates. That [14C]isoleucine became incorporated into insulin and [3H]tryptophan became incorporated into glucagon was established by determination of the distribution of radioactivity in polyacrylamide gels following electrophoresis of labeled proteins recovered from the insulin and glucagon containing portions of gel filtration eluates. These results provide preliminary evidence for sequential metabolic cleavage of proglucagon in glucagon biosynthesis.

Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA.

Single-stranded fragments of adenovirus type 2 DNA were isolated from infected KB cells under conditions which retarded reassociation of complementary sequences but did not denature native viral DNA. Of the total intracellular, virus-specific DNA labeled during a 1-h pulse with tritiated thymidine begining 15 h after infection, about 20% was single stranded when fractionated on hydroxylapatite. This DNA shifted predominantly to the double-stranded fraction on hydroxylapatite during an extended chase incubation, suggesting that it may represent single-stranded DNA in replicating intermediates. Furthermore, the single-stranded DNA annealed nearly equally to both strands of the adenovirus genome. These findings indicate that at least portions of both complementary strands of adenovirus type 2 DNA are exposed as single strands during the period of viral DNA synthesis.

Rapidly labeled intermediates in DNA replication in higher plants

In detached Vicia embryos, the incorporation of [ 3 H]thymidine into DNA starts at about 25 h after the beginning of imbibition and reaches maximum at about 33 h. The DNA synthesized during the first replication phase was extracted. Alkaline sucrose density-gradient analyses of the DNA indicated the occurrence of several short pieces of rapidly labeled DNA having sedimentation values of approx. 10 S and 14 S, after a pulse for 5 to 10 min. Prolonged labeling and chase incubation led to a shift of the shorter fragments to longer ones of 19 S and 22 S or more, thus indicating the nature of intermediates during DNA replication of these short fragments.

Half‐Life of sRNA from Primary Roots of Zea mays. A Contribution to the Cytokinin Production

AbstractRoots produce cytokinins, which could be generated from the catabolism of transfer RNA. To prove such a hypothesis, the half‐life of sRNA from primary roots of corn was measured. The shortest half‐life was estimated to be 3 days. Depending on growth, cells at the root tips are “moving” out of the growing region and become differentiated cells. Therefore the highest label at the root tip during the pulse incubation is moving backwards during the chase incubation. Prolonged chase increases the half‐life of sRNA successively, possibly due to decreasing metabolic activities of the originally labeled cells.

Studies on in vitro synthesis and secretion of growth hormone and prolactin. II. Evidence against the existence of precursor molecules.

In order to establish the possible existence of hormone precursors, the biosynthesis of rat growth hormone. (GH) and prolactin (PRL) was investigated in pituitary slices pulselabeled for a short time (5 min) with 14C-Lleucine and then incubated in chase medium up to 40 min. Proteins were analyzed by two different techniques of gel electrophoresis (the nondissociating gel system of Ornstein and Davis and a high resolution SDS gel system) and the rate of incorporation of 14C-L-leucine and the evolution of the specific radioactivity with chase in total protein as well as in GH and PRL and in other protein bands were followed. No kinetics of hormone labeling compatible with the existence of precursors was ever obtained when samples were adequately solubilized with detergents prior to electrophoresis on either system. Hormone bands appeared highly labeled at the end of the pulse and their specific radioactivity did not change significantly during chase incubation. Only when homogenates were incompletely solubi...

Thyroglobulin synthesis and intracellular transport studied in bovine thyroid slices

The purpose of the study was to elucidate the interrelation between the synthesis of the protein portion of thyroglobulin and the transport of the protein from the endoplasmic reticulum of the follicle cell to the follicle lumen. Bovine thyroid slices were labeled by 20–30 minutes' incubation with leucine-14C and further incubated in a chase medium for up to 4 hours. Four microsomal subfractions and a supernatant fraction were prepared. Labeled proteins were extracted from the fractions and separated by sucrose density gradient centrifugation. The morphology of the slices and the cell fractions was studied by light and electron microscopy. After incubation, labeled proteins were found in a microsomal subfraction consisting of ribosome-studded vesicles (Ms I) and, in smaller amounts, in a subfraction mainly containing smooth-surfaced vesicles (Ms II). In Ms I labeling appeared both in the thyroglobulin region of the density gradient and in “lighter” regions whereas in Ms II labeling was mainly restricted to the thyroglobulin region. During the first hour of chase incubation the labeling decreased in Ms I and increased in Ms II, resulting in inverted relations between the specific radioactivities of the two fractions. This is interpreted to represent a transfer of newly synthesized proteins from the components of Ms I to those of Ms II, which, at the level of the follicle cell, could correspond to a transfer from the cisternae of the rough-surfaced endoplasmic reticulum to apically located, dense vesicles. During the third and fourth hours of chase incubation the specific radio-activity of Ms II declined. In view of the labeling patterns of the other cell fractions and previous in vivo observations, it is suggested that this decline might represent a transfer of protein from the components of Ms II to the colloid portion of the supernatant.

Effects of Selected Drugs on Pancreatic Macromolecular Transport

Abstract It is well known that pancreatic digestive proteins are synthesized on ribosomes attached to endoplasmic reticulum and that nascent proteins are transported through membranes of the endoplasmic reticulum into intracisternal spaces and thence into Golgi apparatus where condensation and encapsulation occur. Digestive proteins are stored in zymogen granules and secreted into pancreatic ductules after a secretory stimulus. This study was designed to examine effects of certain drugs on pancreatic protein synthesis, intracellular transport, and secretion. For these studies, pooled rat pancreatic slices were pulse-labeled with L-phenylalanine- 14 C and migration of the 14 C-labeled proteins studied by chase incubation for 15, 30, 60, and 120 min. After in vitro incubation, subcellular particles were isolated by density gradient centrifugation and specific activity of radioactive-labeled proteins determined in subcellular fractions. These studies indicate that bethanechol chloride did not alter the rate of transport of synthesized proteins from ribosomes into zymogen granules but increased the rate of secretion of proteins from zymogen granules into medium. Tetracycline and amphotericin B impaired transport of labeled proteins from microsomes to zymogen granules as well as secretion of labeled proteins from zymogen granules into medium. Puromycin did not alter either rate of transport of labeled proteins or secretion of labeled proteins into medium. Lidocaine increased appearance of unlabeled proteins into medium but significantly impaired secretion of labeled proteins. These studies provide information concerning fundamental mechanisms involved with transport, synthesis, and secretion of pancreatic digestive proteins.

Biosynthesis of myelin lipids of cultured nervous tissues--incorporation of choline and CDP-choline into myelin phospholipids.

Myelination of cultured brain tissue from 1- to 4-day-old rats was biochemically studied by using radioactive materials. Explants were maintained in agar media by means of organ culture to produce a rather large amount of myelin in vitro required for lipid analysis. Pulse incubation with isotopes was carried out after 2 to 4 weeks in culture. Lipids of myelin and non-myelin fractions isolated from homogenates of explants without or with a fresh rat brain as a “carrier of myelin” were analyzed after chase incubation for 2 to 24 hours. Choline and CDP-choline were actively incorporated during myelination in organ culture into lecithin and sphingomyelin of myelin and non-myelin fractions, radioactivity of which was increased in course of chase incubation time. These results were compared with those of in vivo myelination of young rats.

Synthesis and secretion of growth hormone in the rat anterior pituitary: I. the intracellular pathway, its time course and energy requirements

ABSTRACT The intracellular processes involved in synthesis, transport and storage of newly synthesized proteins in the rat somatotroph, together with their time course and metabolic requirements, have been investigated in a quantitative electron-microscopic radioautography study of the tissue following pulse labelling with tritium-labelled amino acids and chase incubations in various conditions. Proteins are synthesized initially on the rough-surfaced elements of the endoplasmic reticulum and are transported within 10 min after their synthesis to transitional areas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer to the Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after synthesis, while formation of mature storage granules occurs within 2 h following protein synthesis. Further experiments utilizing cycloheximide or ouabain during the chase incubations showed that the intracellular transport of newly synthesized protein and its time course are not significantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassium dependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 μM 2,4-dinitrophenol) or of respiration (10 μM antimycin A) markedly reduced intracellular ATP levels and inhibited the intracellular transport processes. The requirement for ATP appeared to be operative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in the formation of storage granules; possible roles of ATP in these processes are discussed.

Characteristics of DNA synthesis following ultraviolet light irradiation in mouse L cells: Postreplication repair

Mouse L cells partially synchronized as to DNA synthesis were tested for ability to bypass UV-induced DNA photoproducts during postirradiation DNA synthesis. The amount of 3H-thymidine incorporated into the DNA of irradiated cells indicates that dimers remaining unexcised in the DNA, block DNA synthesis, though not permanently. Alkaline sucrose sedimentation has revealed that daughter DNA newly synthesized for 2 h in irradiated cells is discontinuous due to a gap presumably opposite each dimer in parental DNA. Such smaller daughter strands exhibit a dose-dependent reduction in the rate of sedimentation and differ from normally replicating short segments. Taking into account the replication rate reduced by UV irradiation, these gaps in daughter DNA seem to be almost closed by the time the synthesis of the involved DNA units is completed, as the sedimentation rate of daughter DNA approaches that of parental DNA during the subsequent chase incubation of irradiated cells. These results provide evidence for the bypass of photoproducts remaining unexcised in DNA, by which mouse L cells may recover.

Studies on in vitro synthesis and secretion of growth hormone and prolactin. I. Hormone pulse labeling with radioactive leucine.

Rat pituitary slices were incubated in vitro for 5 min in a medium containing trace amounts of 14C—L—leucine of high SA and then reincubated for various times in non—radioactive medium containing an excess of 12C—L—leucine (chase medium). Under these conditions a true pulse labeling of pituitary protein can be obtained since the incorporation of the radioactive amino acid (a) is linear for 5 min and (b) ceases upon transfer of the slices to chase medium. Pulselabeled GH and PL appear in the media only after 45 min of chase incubation; such a lag is regarded as the time needed for the newly synthesized hormones to become available for discharge, i.e., to be transferred from the site of synthesis (the RER) to secretion granules. Once started the release of GH and PL proceeds exponentially for at least 300 min of chase incubation, when -30% of GH and -70% of PL appear to have been discharged. The rate constant of the release processes was found to be 0.055 and 0.49 for GH and PL, respectively. In conclusion,...

Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit.

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2-2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-(3)H, followed by a chase incubation, showed that the label is initially located (chase: 1-6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16-36 min), condensing vacuoles (chase: 36-56 min), immature granules (chase: 56-116 min), and finally mature storage granules (chase: 116-356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.

Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells.

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-(3)H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

Studies of intermediary metabolism in radish cotyledons. Turnover of photosynthetic products in 14CO2 pulse-chase experiments

Ability of 11-day-old radish cotyledons to carry out photosynthesis has been examined by supplying 14CO2 to cotyledon disks for periods ranging from 30 s to 20 min. Carbon-14 was rapidly incorporated into malate, citrate, sugar phosphates, and a number of amino acids. The kinetics of 14C incorporation suggest that carbon initially enters components of the organic acid and sugar phosphate fraction, being then rapidly transferred to the amino acids and sugars. In pulse-chase experiments, illuminated cotyledon disks were incubated with 14CO2 for 30 min then transferred to air containing 12CO2 for periods up to 240 min with illumination. During such postpulse incubations, considerable losses of 14C occurred from most of the principal compounds labeled during the chase incubation. As these losses were not entirely accounted for by losses of label to the incubation medium they can largely be attributed to metabolic turnover of these compounds. It is concluded that, in general, the photosynthesis of radish cotyledons is similar to that of mature leaves and of physiological significance as a route for synthesis of several important cellular constituents required for seedling growth and development.

Radioautographic studies of choline incorporation into peripheral nerve myelin.

This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.

Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex.

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-(14)C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.

Synthesis of messenger ribonucleic acid in excised pea-seedling root segments. Separation of the messenger from microsomes by electrophoresis

1. Electrophoresis on cellulose acetate membrane in a tris-pyrophosphate buffer was used to separate microsomal fractions into three components: (1) the lipoprotein; (2) the nucleoprotein (termed the beta-band); (3) traces of free RNA (termed the alpha-band). In tris buffer containing Mg(2+) the alpha-band was not obtained. 2. The incorporation of uridine and phosphate into RNA by excised pea-seedling root segments was studied by using this electrophoretic technique. 3. It was shown that after a short (;pulse') incubation in the radioactive precursor and a longer (;chase') incubation in the non-radioactive precursor most of the incorporation was into the RNA of the alpha-band and little into that of the beta-band. Previous work showed that in roots of whole seedlings the incorporation is mostly into the ribosomal RNA, corresponding to the material in the beta-band. 4. A pulse-labelled RNA has also been found; this seems to be a cell fraction distinct from the microsomes or ribosomes. 5. The apparent base compositions of labelled RNA in the alpha-band and small amounts of labelled RNA in the beta-band and of unfractionated RNA were very different from the composition of ribosomal or transfer RNA, and somewhat like that of DNA. 6. It is suggested that the excised root segment synthesizes a messenger-RNA fraction labelled after a pulse incubation and a distinct messenger RNA labelled after a pulse and chase incubation, but no ribosomal or transfer RNA. The system is thus similar to the ;step-down' culture conditions in bacteria.