Secretion of β-glucosidase by Ochromonas danica

-Glucosidase released by the phytoflagellate Ochromonas danica was the result of secretion; this was adduced from the following: (1) The enzyme was released during growth, including early log phase. (2) The amount released was calculated to be much more than could be attributed to cell lysis. (3) -Glucosidase was released by cells during short term incubation in a dilute salt solution; this release was nearly linear for at least 24 h. (4) Release occurred while cell counts remained nearly constant and cells remained viable. (5) Control experiments excluded cell damage resulting from incubation and cell manipulation as a source of the exoenzyme. (6) No alkaline phosphatase was released and 5 times less phosphoglucose isomerase than glucosidase was released while the cells contained 7 times more phosphoglucose isomerase. (7) The kinetics of release of nonspecific protein and UV absorbing material was markedly different from glucosidase release. (8) Glucosidase release was temperature and energy dependent; anaerobiosis decreased enzyme release. (9) Release was inhibited by cycloheximide. (10) Cells incubated with ³H-leucine synthesized labeled protein which was secreted linearly for at least 24h. Cycloheximide inhibited incorporation of ³H-leucine into protein and the secretion of the labeled protein.Non-Standard Abbreviations CHI cycloheximide - DNP 2,4-dinitrophenol - IAA iodoacetic acid - PGI phosphoglucose isomerase - SIS salt incubation solution     

Program of early development in the mammal: Synthesis and intracellular migration of histone H4 during oogenesis in the mouse

Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modified high-resolution two-dimensional gel electrophoresis procedure capable of resolving basic proteins (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol.73, 103–119). Histones were separated on such gels and observed rates of incorporation of [³⁵S]methionine into histone H4 were converted into absolute rates of synthesis by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and unfertilized eggs Schultz et al., 1979a, Schultz et al., 1979b. Histone H4 was synthesized at all stages of oogenesis examined, and accounted for 0.07, 0.05, and 0.04% of total protein synthesis in growing oocytes, fully grown oocytes, and unfertilized eggs, respectively. During oocyte maturation the absolute rate of histone H4 synthesis decreased by about 40%, as compared to a 23% decrease in the rate of total protein synthesis during the same period. These measurements indicate that enough histone is synthesized during oogenesis in the mouse to support two to three cell divisions. Examination of the intracellular location of newly synthesized proteins in fully grown oocytes revealed that histone H4 was highly concentrated in the nucleus (germinal vesicle), whereas total protein and tubulin were not. Nearly 50% of the histone H4 synthesized during a 5-hr period was located in the oocyte's germinal vesicle, as compared to 1.9 and 0.9% for total protein and tubulin, respectively. These results are compared with those obtained using oocytes and eggs from nonmammalian animal species.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Synthesis and secretion of salivary gland proteins in Drosophila gibberosa during larval and prepupal development

Summary The late larvae of Drosophila gibberosa Patterson and Mainland choose different pupariation sites than the larvae of Drosophila melanogaster Meigen. Since the larvae of D. gibberosa do not attach themselves to the substratum, the salivary glands contain only a small amount of the glue proteins before pupariation. Proteins comprising the salivary gland secretions of late larvae of these two species were compared and found to be qualitatively quite different. Only five polypeptides with the same molecular masses were identified in both species. The rate of protein synthesis in the salivary glands of D. gibberosa continued to increase through the late larval stage and pupariation. As a consequence, the total amount of protein contained in the salivary glands also continued to increase after pupariation. To demonstrate temporal changes in protein synthesis from 48 h before pupariation to 28 h after pupariation, newly synthesized polypeptides were pulse labeled by culturing salivary glands in vitro. The patterns of polypeptide synthesis fell into four major groups depending upon whether the synthesis of a protein stopped shortly after pupariation, stopped during late pupariation, increased at pupariation, or was initiated after pupariation. Changing patterns of protein synthesis are correlated with the known changes in gene puffing during this developmental period.     

Injected mRNA does not increase protein synthesis in unfertilized, fertilized, or ammonia-activated sea urchin eggs

We have investigated whether the rate of protein synthesis in unfertilized and fertilization-activated sea urchin eggs is limited by the availability of mRNA by injecting eggs, zygotes, and ammonia-activated eggs with globin mRNA. Message-injected and buffer-injected cells were labeled with radioactive amino acids and the proteins separated on a polyacrylamide gel. The relative amounts of newly synthesized globin and endogenous proteins were obtained by scanning the gel fluorograph. Globin mRNA is translated poorly in Strongylocentrotus droebachiensis eggs and does not significantly increase or decrease endogenous protein synthesis. In zygotes and ammonia-activated eggs, however, globin mRNA is translated well and appears to compete with endogenous mRNAs for the limiting component of the translational machinery as it is released. Our results are consistent with the hypothesis that either ribosomes or recruitment factors are gradually activated after fertilization or ammonia treatment, that such components are the rate-limiting factor, and that they impart the typical sigmoidal increase in protein synthesis rate observed in fertilized eggs before the first cleavage.     

Cell-specific regulation of protein synthesis in the sea urchin gastrula: A two-dimensional electrophoretic study

Protein synthesis in the two cell types of echinoid early gastrulae was analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Epithelial cells and primary mesenchyme cells were isolated from early gastrulae as described by M. A. Harkey and A. H. Whiteley, 1980 (Wilhelm. Roux's. Arch.189, 111–112). Newly synthesized proteins were labeled with [³H]valine, extracted in SDS buffer, and analyzed electrophoretically. Of the 454 labeled proteins analyzed, 58 incorporated [³H]valine at markedly different relative rates in the two cell types, and 69 were labeled exclusively in one or the other cell type. The most rapidly synthesized proteins in gastrula cells constituted a class which exhibited a much higher degree of cell specificity than the total protein population. Several of these rapidly synthesized proteins were analyzed individually. Among those that were synthesized preferentially in primary mesenchyme cells, two low-molecular-weight, acidic proteins, designated PM28 and PM32, accounted for 9–14% of the total protein synthesis in primary mesenchyme cells but were barely detectable in epithelial cells. Those proteins that were synthesized preferentially in the epithelial cells included several low-molecular-weight species, probably histones, and the cytoskeletal proteins, actin and tubulin. These data indicate that the primary mesenchyme and epithelium of the early gastrula differ profoundly with respect to the synthesis of specific proteins.     

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This “burst phase” lasts about 3–5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol.40, 245–255; D. Epel, 1978, Curr. Top. Dev. Biol.12, 185–246) that initiation of endocytosis is most likely a Ca²⁺-dependent event.     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
• 2.2. When cells were incubated with [2,8-³H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
• 3.3. Upon incubation of cells with [α-³²P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, ³²P-labeling of the 26 kDa protein was observed.
• 4.4. After treatment with snake venom phosphodiesterase, [³²P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-³²P]ATP.
• 5.5. The ³²P-labeling pattern of proteins with [α-³²P]ATP was clearly different from that with [adenylate-³²P]NAD⁺.
• 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.

     

Major oligomeric structural proteins of the HeLa nucleus

The three predominant polypeptides of the insoluble proteinaceous fraction from the HeLa cell nucleus polymerize in vitro upon oxidation of intrinsic sulfhydryl groups. The ease and specificity of this reaction indicate that these polypeptides exist as ordered oligomers in vivo. The comparable insoluble fraction from the rat liver nucleus also contains three predominant polypeptides of the same molecular weights, 65,000 71,000, and 75,000. The insoluble protein of the avian erythrocyte nuclear envelope consists principally of the 71,000- and the 75,000-dalton polypeptides. Indeed, in the avian erythrocyte nucleus these are the predominant polypeptides of the entire nucleus (Shelton, K., Cobbs, C., Povlishock, J. and Burkat, R., 1976, Arch. Biochem. Biophys.174, 177). Further, these avian polypeptides each form homogeneous covalently linked oligomers upon sulfhydryl oxidation (Cochran, D., Cobbs, C. and Shelton, K., 1977, J. Cell Biol.75, 151a). The insolubility, oligomeric disposition, and relative prominence of these polypeptides in a wide variety of cells indicate a fundamental structural role in the nucleus. Morphological features which may reflect this structural or skeletal role could be the nuclear envelope, the fibrous lamina, or perhaps an intrachromatinic matrix. The metabolism of the oligomeric polypeptides has been investigated in HeLa cells. Turnover of the HeLa insoluble nuclear protein is similar to that of the histones which are known to be stable proteins. The insoluble protein, including the oligomeric polypeptides, is synthesized in G1, S, and G2 phases of the cell cycle. This metabolic behavior indicates that the oligomeric polypeptides are reutilized in successive cell cycles and that synthesis accompanies nuclear and cellular expansion rather than deoxyribonucleohistone synthesis. This suggests that neither degradation nor selective synthesis of oligomeric polypeptides at a particular phase of the cell cycle are responsible for the breakdown and reformation of the interphase cell morphological features that occur during mitosis.     

Human fibroblast-conditioned medium contains a 100K dalton glucose-regulated cell surface protein

This report describes the identification and partial characterization of a 100K dalton “glucose-regulated” cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (“conditioned medium”) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.     

Synthesis of unique proteins at the onset of carbon starvation inEscherichia coli

Summary Escherichia coli bulk protein synthesis continued during the first 3–4 h of carbon starvation at 50–75% that of non-starved (growing) cells. Two-dimensional gel electrophoresis analysis of in vivo pulse-labelled proteins resolved at least 30 polypeptides with new or increased synthesis, relative to total protein synthesis, during this time. Among these polypeptides were several that were also synthesized by ethanol-treatedE. coli (heat-shock proteins). In addition, a number of unique polypeptides were synthesized by carbon-starved cells. These starvation proteins may be involved in survival of the starving bacteria.     

The synthesis of hemolymph proteins by the larval midgut of an insect Calpodes ethlius (Lepidoptera:Hesperiidae)

Ligated tubes of Calpodes ethlius (Lepidoptera:Hesperiidae) larval midgut with normal (i.e. apical secretions into the lumen and basal into the hemocel or medium) or inverted orientation (i.e. apical secretions into the hemocoel or medium and basal into the lumen) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The midgut synthesizes and secretes at least eight polypeptides basally and seven apically. The tissue also synthesizes many other polypeptides that are not released at either surface. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) proteins synthesized in vitro are the same as those synthesized in vivo, (b) different proteins are secreted on apical and basal surfaces, (c) in vitro apical secretions are the same as in vivo luminal proteins, (d) at least two of the basal secretions can be demonstrated in the hemolymph labeled in vivo. Almost all basal secretions showed immunological similarity with hemolymph proteins as observed by immunoprecipitation and fluorography. Arylphorin is a main hemolymph protein synthesized by the midgut. Midgut arylphorin has been identified by its precipitation by antibodies to hemolymph arylphorin. We conclude that insect midgut has bi-directional secretion. Luminal proteins (presumably digestive enzymes and perhaps goblet cell luminal contents) are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Thrombin induced surface changes of human platelets.

Membrane proteins of both control and thrombin-treated human platelets were labeled by (³H)-sodium borohydride reduction of Schiff bases formed between pyridoxal phosphate and protein amino groups. Fluorographic analysis of solubilized platelet proteins disclosed a substantial difference between the labeling patterns of control vs. thrombin-treated cells. Whereas the normal platelets showed a single intensely labeled protein band, cells exposed to thrombin showed about ten labeled polypeptides. When thrombin-induced serotonin release from platelets was blocked by 3′,5′-adenosine diphosphate, the protein labeling pattern on the fluorograph resembled that of control platelets. These data suggest that serotonin release from platelets by thrombin involve major changes in the architecture of proteins of platelets surface.     

G1 and S phase mammalian cells synthesize histones at equivalent rates

To determine the effect of cell cycle position on protein synthesis, synchronized cell populations were metabolically labeled and the synthesis of the basic proteins, including histones, was examined by two-dimensional gel electrophoresis. Exponentially growing S49 mouse lymphoma or Chinese hamster ovary (CHO) cells were separated into G1 and S phase populations by centrifugal elutriation, selective mitotic detachment, fluorescence-activated cell sorting, or a combination of these, and pulse-labeled with radiolabeled amino acids. The histone proteins, both free and chromatin-bound, were completely resolved from some 300 other basic polypeptides in whole-cell lysates by a modification of the NEPHGE technique of O'Farrell, Goodman and O'Farrell (1977). Comparisons of matched autoradiograms from samples of G1 and S phase labeled cells revealed an equivalent rate of histone synthesis through the cell cycle of both S49 and CHO cells. Nuclei isolated from G1 phase S49 cells that were pulse-labeled contained between 13 and 15% of the newly synthesized nucleosomal histones present in S phase nuclei. Nuclei prepared from G1 phase cells that were pulse-labeled and then chased for 5 hr contained more than 90% of the labeled nucleosomal histones present in wholecell lysates. It therefore seems likely that differential alterations in the rate of histone synthesis do not occur to a significant degree as cells proceed through the cycle, but the association of newly synthesized histones with DNA takes place after the onset of DNA replication.     

The effect of water loss upon the urate,urea and ammonia content of the egg of the Japanese quail Coturnix coturnix japonica

• 1.1. The urate, urea and ammonia content of the whole egg of the Japanese quail was measured in late incubation in eggs subject to different rates of water loss.
• 2.2. High rates of water loss substantially increased egg urate content, but had little or no effect on urea or ammonia content.
• 3.3. Allopurinol, an inhibitor of urate synthesis, reduced egg urate content to low levels, but produced no effect on urea content, and a small reduction in ammonia content.
• 4.4. The urea concentration of the embryo was lower than in allantoic fluid.
• 5.5. It is concluded that urate production by the avian embryo is primarily concerned with the modification of allantoic fluid composition.

     

Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2

The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem.256, 1604–1607, as adapted for photosynthetic membranes by T. M. Bricker and L. A. Sherman (1982) FEBS Lett.149, 197–202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b₆, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c₅₅₀ and c₅₅₄, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.     

Water-soluble Ca2+-calmodulin-binding proteins in embryo of the sea urchin Strongylocentrotus intermedius

• 1.1. About 0.3–0.4% of total water-soluble protein extracted from sea urchin embryos at the two-cell and early-gastrula stages was Ca²⁺-dependently bound to immobilized calmodulin.
• 2.2. SDS-PAGE of calmodulin-binding proteins revealed at least 20 polypeptides ranging from 200 to 15.5 kDa, and 70–80% of the protein belonged to a dozen major polypeptides. Polypeptides of 70, 55, 50, 45 and 18 kDa seemed to be the same as those that were detected earlier (Iwasa and Mohri, J. Biochem.94, 575–587, 1983).
• 3.3. The polypeptide spectrum of calmodulin target proteins changed significantly, e.g. the major polypeptides of 70 and 41 kDa increased, and the 200 and 43 kDa polypeptides decreased sharply during development from the two-cell to the early-gastrula stage.
• 4.4. According to our estimates, the molar concentrations of the calmodulin and targets were close enough, and therefore the Ca²⁺ signal should depend on the spatial-temporal distribution of free calmodulin in the cells.

     

Endogenous ammonia production by Anacystis nidulans R-2 induced by methionine sulfoximine

Anacystis nidulans R-2 produced ammonia from endogenous sources for at least 6 h when illuminated without external nitrogen source but with CO₂ in the presence of 50 M methionine sulfoximine. The onset of ammonia release coinciding with complete inhibition of glutamine synthetase. The total quantity of ammonia which could be released exceeded the nitrogen content of small molecule pools, and suggested protein degradation as the most likely source of the nitrogen. Ammonia release was not accompanied by leakage of carbon compounds from the cells. Methionine sulfoximine-induced ammonia release was energy requiring, and was barely detectable under dark anaerobic conditions, or in the presence of 10 M carbonyl cyanide m-chlorophenylhydrazone in light. Phenyl methyl sulfonylfluoride, an inhibitor of serine proteases, eliminated ammonia release, and the rate of release was reduced to one-third of control values, after a lag, in the presence of 50–75 g/ml chloramphenicol. The rate of NH ₊ ⁴ release was maximal (1.4 nmol·min^-1·mg^-1 protein) if suspensions were bubbled with 100% O₂, but could not be reduced below 0.6 nmol·min^-1·mg^-1 protein in air: CO₂, suggesting that release was at most only partly due to photorespiration.Abbreviations used MSX L-methionine D,L-sulfoximine - PMSF phenylmethylsulfonylfluoride - CAP chloramphenicol - CCCP carbonyl cyanide-m-chlorophenyl hydrazone