Immunocytochemical analysis of protein body formation in seeds of Sorghum bicolor

1989 ◽  
Vol 67 (10) ◽  
pp. 2850-2856 ◽  
Author(s):  
Hari B. Krishnan ◽  
Jerry A. White ◽  
Steven G. Pueppke
Keyword(s):  
Endoplasmic Reticulum ◽  
Sorghum Bicolor ◽  
Rough Endoplasmic Reticulum ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Protein Body ◽  
Electrophoretic Analysis ◽  
Protein Bodies ◽  
Aleurone Layer ◽  
Dark Staining

Electrophoretic analysis of sorghum (Sorghum bicolor (L.) Moench) seed prolamines in the presence of sodium dodecyl sulfate reveals major proteins of 27 and 25 kDa and two other proteins of 18 and 12 kDa. Antibodies were raised against this prolamine fraction and used to examine the subcellular distribution of the proteins in developing sorghum seeds. Protein bodies in the starchy endosperm and subaleurone cells usually are round in cross section and contain darkly staining materials arranged in concentric rings. Protein bodies in the first two layers beneath the aleurone layer are irregular in shape and contain discrete pockets of light and dark staining inclusions. Prolamines were detected in both types of protein bodies by immunolabeling. Other oganelles, including Golgi complexes, mitochondria, and amyloplasts, were not labeled. The protein bodies, which have ribosomes attached to their surfaces, are directly connected to the rough endoplasmic reticulum. In some instances, this endoplasmic reticulum was specifically labeled with protein A – gold particles. Based on these observations, we suggest that the rough endoplasmic reticulum serves as the site of both synthesis and accumulation of sorghum prolamines.

Seasonally dependent formation of protein-storage vacuoles in the inner bark tissues of Salix microstachya

1990 ◽  
Vol 68 (8) ◽  
pp. 1747-1755 ◽  
Author(s):  
John S. Greenwood ◽  
Cobi Demmers ◽  
Suzanne Wetzel
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Protein Body ◽  
Protein Bodies ◽  
Electron Microscopic ◽  
Protein Storage ◽  
Inner Bark ◽  
Protein Storage Vacuoles ◽  
Body Development ◽  
Microscopic Studies

The inner bark tissues of temperate hardwoods often act in the temporary storage of reduced nitrogen as protein during the overwintering period. Electron microscopic studies reported here demonstrate the analogy between the protein-storage vacuoles of the inner bark tissues and protein bodies in seeds. Development of these organelles parallels that of protein body formation seen in many dicotyledonous seeds. Coincident with the synthesis and sequestering of specific proteins, the large central vacuoles of the phloem parenchyma cells are slowly replaced over a 3- to 4-week period with numerous smaller protein-storage vacuoles (protein bodies). These arise via the subdivision of the larger vacuole and subsequent filling of the smaller vacuoles with protein. During this process there is a proliferation of both free ribosomes and rough endoplasmic reticulum in the ground cytoplasm. Stacks of rough endoplasmic reticulum are present in the peripheral cytoplasm and surround the smaller vacuoles as proteinaceous material is deposited. Golgi complexes, although not numerous, are present in the ground cytoplasm during the filling of the protein storage vacuoles. Key words: protein-storage vacuoles, protein body development, Salix microstachya, hardening, nitrogen storage, dormancy onset.

Preliminary Treatment of Urinary Proteins Improves Electrophoretic Analysis and Immunodetection

1992 ◽  
Vol 38 (6) ◽  
pp. 860-863 ◽  
Author(s):  
J M Verdier ◽  
B Dussol ◽  
P Dupuy ◽  
Y Berland ◽  
J C Dagorn
Keyword(s):  
Protein Pattern ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Complex Mixture ◽  
Electrophoretic Analysis ◽  
Renal Physiology ◽  
Preliminary Treatment ◽  
Urinary Proteins ◽  
Simple Method ◽  
Electrophoretic Migration

Abstract Analysis of urinary protein composition is an important tool in studies on renal physiology and physiopathology. Urine is, however, a complex mixture containing, besides protein, a variety of compounds such as salts, peptides, oligosaccharides, and glycosaminoglycans. Some of these compounds interfere with the electrophoretic migration of protein in sodium dodecyl sulfate-polyacrylamide gels and prevent correct analysis of the protein pattern. We describe a simple method for extracting urinary proteins that considerably improves their electrophoretic migration and subsequent immunodetection. This treatment involves ammonium sulfate fractionations (for precipitating proteins), EDTA (for inhibiting protein aggregation), and HCl hydrolysis (for removing glycosylaminoglycans). Recovery during extraction was found to be almost quantitative for total protein and three representative proteins: albumin, alpha 1-glycoprotein acid, and beta 2-microglobulin.

Immunocytochemical localization of wheat prolamins in the lumen of the rough endoplasmic reticulum

1991 ◽  
Vol 69 (11) ◽  
pp. 2574-2577 ◽  
Author(s):  
Hari B. Krishnan ◽  
Jerry A. White ◽  
Steven G. Pueppke
Keyword(s):  
Triticum Aestivum ◽  
Endoplasmic Reticulum ◽  
Key Words ◽  
Rough Endoplasmic Reticulum ◽  
Protein A ◽  
Triticum Aestivum L ◽  
Soluble Proteins ◽  
Immunocytochemical Localization ◽  
Specific Antibodies ◽  
Days After Anthesis

Antibodies raised against gliadins, the alcohol-soluble proteins of wheat (Triticum aestivum L.) seeds, were used to localize gliadins within the lumen of the endoplasmic reticulum. Endosperm cells at 20 days after anthesis contain extensive rough endoplasmic reticulum that is fragmented and dilated. The dilated endoplasmic reticulum encloses aggregates of proteinaceous material that reacts strongly with gliadin-specific antibodies. Key words: gliadins, immunocytochemistry, protein A – gold, rough endoplasmic reticulum, wheat.

scholarly journals Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

1989 ◽  
Vol 262 (2) ◽  
pp. 497-503 ◽  
Author(s):  
J Lanoix ◽  
L Roy ◽  
J Paiement
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Electrophoretic Analysis ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Dog Pancreas

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5′-[gamma-thio]triphosphate or guanosine 5′-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.

scholarly journals Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

1985 ◽  
Vol 101 (6) ◽  
pp. 2355-2365 ◽  
Author(s):  
A Ratcliffe ◽  
P R Fryer ◽  
T E Hardingham
Keyword(s):  
Endoplasmic Reticulum ◽  
Chondroitin Sulfate ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Localization ◽  
Protein A ◽  
Keratan Sulfate ◽  
Glycosaminoglycan Synthesis ◽  
Protein Core ◽  
Late Event ◽  
Secretory Glycoproteins

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Ébauches intracytoplasmiques de grains protéiques dans des cotylédons de Raphanus en développement

1986 ◽  
Vol 64 (12) ◽  
pp. 2887-2895
Author(s):  
Louis Genevès ◽  
Jacques Rutin
Keyword(s):  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Rough Endoplasmic Reticulum ◽  
Early Phase ◽  
Acid Phosphatase Activity ◽  
Early Stage ◽  
Protein Bodies ◽  
Meristematic Cells ◽  
Size And Structure

Protein bodies were characterized at an early stage of their maturation in thin green cotyledons of developing radish embryos. They appeared as granules in the cytoplasm of meristematic cells. Their diameter (0.5 to 1 μm) was in the range of that of mitochondria. They were distinguished from vacuoles by their morphology, size, and structure. Some appeared to be associated with cisternae of endoplasmic reticulum or dictyosomes (permanganic fixations). Their evolution was synchronous in the cell and also in the cotyledonary tissue. Compact in appearance, they were constituted of thin packed fibrillar structures, limited by a denser peripheral layer. It is difficult to know whether or not they had a limiting membrane. Some possessed thin dense crystals or globoids (aldehydic fixations). During this early phase, several types of organelles seemed to contribute to the development of protein bodies, including saccules of rough endoplasmic reticulum and dictyosomes. Polyribosomes constituted a network around their surface. They did not exhibit any acid phosphatase activity. In this respect, they differed from the vacuoles, saccules of endoplasmic reticulum, and several neighbouring vesicles.

scholarly journals Quantitative immunocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell.

1980 ◽  
Vol 28 (2) ◽  
pp. 149-160 ◽  
Author(s):  
M Bendayan ◽  
J Roth ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Protein A ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Thin Sections ◽  
Intracellular Compartments ◽  
Rat Exocrine Pancreas ◽  
Acinar Lumen ◽  
Cellular Compartments

The recently developed protein A-gold technique for the detection of intracellular antigenic sites on thin sections was utilized to localize nine different secretory proteins in the rat exocrine pancreas. Amylase, chymotrypsinogen, trypsinogen, lipase, elastase, carboxypeptidases A and B, RNase and DNase, were detected at the level of the rough endoplasmic reticulum, the Golgi area, and the zymogen granules of the acinar cells, as well as in the acinar lumen. A quantitative evaluation of the labeling showed that its intensity was not identical for all enzymes studied nor in all cellular compartments analyzed. An increasing gradient of the labeling from the rough endoplasmic reticulum to the Golgi and to the zymogen granules was found for amylase, carboxypeptidases A and B, chymotrypsinogen, trypsinogen, and RNase, while a comparable low degree of labeling in the Golgi apparatus and in the zymogen granules was observed for DNase, lipase, and elastase. These results suggest that the nine enzymes are processed through the same intracellular compartments, but that they may be concentrated to different degrees in the zymogen granules before being released in the acinar lumen.

scholarly journals CHANGES IN THE RIBOSOME CONTENT, PRINCIPAL MICROSOMAL PROTEIN COMPOSITION, AND SECRETORY CHARACTER OF MAMMARY EPITHELIAL ROUGH ENDOPLASMIC RETICULUM DURING DIFFERENTIATION

1974 ◽  
Vol 61 (3) ◽  
pp. 613-632 ◽  
Author(s):  
Frank Slaby ◽  
Carolyn Brown
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Mammary Development ◽  
Mammary Epithelial ◽  
Density Range ◽  
Short Period ◽  
Rough Er

The equilibrium density distribution, protein composition, and secretory character of mouse mammary epithelial rough microsomes have been determined during differentiation. The density range exhibited by the rough microsomes broadens during mammary development; rough microsomes within the 1.25–1.29 g/ml density range appear soon after conception and then within the 1.30–1.34 range after the onset of lactation. The appearance of these denser microsomes represents the progressive increase of the average ribosome content of the rough endoplasmic reticulum (ER) during gestation and lactation. Fractionation of rough microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two proteins, having molecular weights of 57,000 and 76,000, occur to a significant extent only during lactation and are then most prominent in the very dense rough microsomes of the 1.30–1.34 range. Nascent polypeptide chains discharged (by incubation with puromycin) from 17-days lactation rough microsomes in either the 1.21–1.29 or 1.30–1.34 density range are distributed equally between the intra- and extravesicular compartments. Whereas 36% of the chains are discharged intravesicularly from 1-day lactation rough microsomes in the 1.30–1.34 range, only 25% are so discharged from those in the 1.21–1.29 range. The results indicate (a) that there is no correlation between the relative levels in lactation rough microsomes of the two microsomal proteins which become prominent during lactation and the extent of secretory activity and (b) that for a short period after parturition the rough ER elements bearing high surface densities of ribosomes have a greater proportion of ribosomes synthesizing milk proteins than the rough ER elements with moderate ribosome densities.

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