Effects of 6-aminonicotinamide on levels of soluble proteins and enzyme activities in various tissues of Japanese quail

The effects of 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of Japanese quail were investigated. SDS-polyacrylamide gel electrophoresis showed that the soluble proteins with molecular masses corresponding to 160.4 and 52.5 kDa were either missing or present at lower concentrations in the brain of the 6-AN treated group compared to those in the control group. The soluble liver proteins with molecular masses 200, 120 and 70.5 kDa were missing in the treated group compared to those in the control while those of a molecular mass 15.1 kDa were found to be present at higher concentrations. Similarly, treatment with 6-AN decreased the concentration of soluble proteins in pectoral muscle with molecular masses 92.3, 54.5, 43.5, 41.2, 34.5, 27.5, 20.1 and 17.5 kDa and increased those with molecular masses 96.5, 37.7, 25.0, 19.3, 16.6, 13.8 and 10.8 kDa. In the heart, soluble proteins with molecular mass 84.6 kDa were increased. There was a marked reduction in the treatment group in the concentration of NAD in pectoral muscle but not in other tissues. A similar observation was also made with total RNA levels. The specific activity of malic enzyme was markedly increased by 6-AN treatment in the kidney and pectoral muscle but reduced in the liver. 6-Phosphogluconate dehydrogenase and lactate dehydrogenase activities were markedly reduced in the liver. Glyceraldehyde-3-phosphate dehydrogenase activity was significantly decreased in liver and pectoral muscle. NAD glycohydrolase activity was markedly decreased in pectoral muscle. Acetylcholinesterase activity was markedly reduced in liver but was enhanced in pectoral muscle. The results suggest that the metabolic actions of 6-AN are specific for certain proteins in the liver and muscle with the effect being most pronounced in muscle. The effects are also quite distinct from those shown by its analogue 3-acetylpyridine.     

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.     

Loblolly pine seed dormancy. I. The relationship between protein synthesis and the loss of dormancy

Embryos from the mature unstratified loblolly pine ( Pinus taeda L.) seeds used in this study were nondormant: however, they failed to germinate in situ because of constraints imposed by the surrounding tissues. During a stratification period of 35 days of moist chilling at 2°C, seed germinability increased from 19 to 76%. The total lipid content of the megagametophyte did not change during stratification, whereas the total protein content of both megagametophyte and embryo was more variable. The rate of synthesis of buffer soluble proteins in these two tissues increased and electrophoretic analysis showed that while similar proteins were synthesized during the stratification period, changes in the patterns of synthesis of some proteins did occur. In both the embryo and megagametophyte the synthesis of a set of proteins with molecular masses below 46 kDa decreased markedly after 14 days of chilling (DOC). In the megagametophyte, the synthesis of a more diverse set of proteins with molecular masses ranging from 16 to 78 kDa increased after 14 DOC. It is noteworthy that these changes in the patterns of protein synthesis coincided with the greatest relative increase in seed germinability of 35%.     

Phosphorylation of low molecular mass cytosolic proteins by protein kinase C and protein kinase A in the rabbit exocrine pancreas

Subcellular fractionation of rabbit pancreatic acini was performed to study the distribution of endogenous substrates for protein kinase C. Substrates for protein kinase C were found to be predominantly low molecular mass proteins of cytosolic origin. At least three of these soluble substrates, with molecular masses of 17-19 kDa, were relatively heavily phosphorylated by endogenous as well as purified pancreatic protein kinase C. In the same molecular mass range, 16-18 kDa, soluble proteins were also phosphorylated by protein kinase A. Moreover, addition of cyclic AMP under conditions that activated protein kinase C gave a more than additive labelling of these low molecular mass proteins. The latter observation may be of interest in view of the potentiating effect cyclic-AMP-activated protein kinase A has on amylase secretion stimulated by secretagogues which increase free cytosolic Ca2+ and activate protein kinase C.     

Effects of neurotoxin 6-aminonicotinamide on levels of enzyme activities and metabolites in quail plasma

Effects of 6-aminonicotinamide (6-AN) on the levels of proteins, metabolites and enzyme activities in the plasma of Japanese quail were investigated. The concentrations of soluble proteins in the pectoral and hindlimb muscle of the 6-AN treated and the pair-fed groups were significantly reduced compared to the control group. In the plasma, the levels of total proteins and albumin were not affected, but the levels of globulin were significantly lower than those of the control and pair-fed groups. In contrast, the levels of glucose and creatine were significantly elevated.Cellulose acetate gel electrophoresis showed that 6-AN induced a new synthesis of prealbumin and also increased the levels of beta-globulin relative to the control and pair-fed groups. In contrast, the levels of gamma-globulin were markedly lower than those of the control group, whereas the levels of alpha-globulin were not affected.The specific activity of alkaline phosphatase of the 6-AN group was significantly lower than that of the control and pair-fed groups and that of aspartate aminotransferase only lower than that of the control group but not the pair-fed group. The specific activities of creatine phosphokinase and lactate dehydrogenase of the 6-AN group were the greatest among the three groups, whereas those of the pair-fed group were greater than those of the control group.The results suggest that 6-AN may interfere with the proper maintenance of energy charges and the immune system function.     

28 kDa adenosine-binding proteins of brain and other tissues.

Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.     

Outer membrane proteins of Fusobacterium nucleatum Fev1

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100 degrees C, respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.     

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree,Sapindus mukorassi,was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis,Western-blot,immuno-histochemical localization,light- and electro-microscopy,together with analysis of proteinase inhibitor activity of the purified VSP in vitro.There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S.mukorassi in leafless periods.The proteins decreased markedly during young shoot development,indicating their role in seasonal nitrogen storage.Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells.The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope.So,the 23 kDa protein was a typical VSP in S.mukorassi.The 23 and 27 kDa proteins shared no immuno-relatedness,whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity.The 23 kDa protein may confer dual functions:nitrogen storage and defense.     

Calmodulin and calmodulin-binding proteins in liver cell nuclei

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.     

Detection of small selenium-containing proteins in tissues of the rat

The important role of selenium in the mammalian organism has been manifested by the detection of several selenoenzymes, and there are still numerous selenium-containing proteins to be identified. After in vivo labeling of rats with [75Se]-selenite, gel electrophoretic separation of the proteins in tissue homogenates and autoradiography of the labeled bands, information on the selenium-containing proteins present in the different tissues was obtained. In the separation by SDS-PAGE and two-dimensional IEF/SDS-PAGE a large number of selenium-containing proteins or protein subunits with apparent molecular masses in the range from 116 to 8 kDa could be distinguished. This range was extended by applying a modified Tricine-SDS-PAGE, which allows the determination of smaller proteins. Using this method in the separation of the homogenates of the adrenal, brain, diaphragm, epididymis, heart, kidney, liver, lung, pituitary, prostate, skeletal muscle, spleen, thymus and thyroid, four additional selenium-containing proteins with molecular masses of approximately 7 kDa, 5kDa, 4 kDa and 3kDa were detected. The 5 kDa protein and the 7 kDa protein were identified as selenocysteine-containing selenoproteins.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.     

Nectarin I is a novel,soluble germin-like protein expressed in the nectar of Nicotiana sp.

We have identified a limited number of proteins secreted into the nectar of tobacco plants. Nectarin I is the most highly expressed nectar protein and has a monomer molecular mass of 29 kDa. The other major nectar proteins are expressed at lower levels and have monomer molecular masses of 41, 54, and 65 kDa respectively. Nectarin I was purified and antiserum was raised against the protein. Under nondenaturing conditions, Nectarin I has an apparent molecular mass of >120 kDa. The expression of Nectarin I was restricted to nectary tissues and to a much lower level in the ovary. No Nectarin I was found in petals, stems, leaves, or roots or other floral tissues. The expression of Nectarin I was also developmentally regulated. It is expressed in nectary tissues only while nectar is being actively secreted. Subsequently, the N-terminus of purified Nectarin I was sequenced. Sequence identity showed Nectarin I is related to wheat germin. Although hydrogen peroxide is readily detectable in tobacco floral nectar, we were unable to demonstrate any oxalate oxidase activity for Nectarin I. A partial cDNA encoding the mature Nectarin I N-terminus was isolated and used to probe a Nicotiana plumbaginifolia genomic library. The Nectarin I gene was isolated and the translated sequence was consistent with both N-terminal and internal cyanogen bromide-derived amino acid sequence. The gene contains a single 386 nt intron and encodes a mature protein of 197 amino acids.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

Insulin-like growth factor I receptors on mouse neuroblastoma cells. Two beta subunits are derived from differences in glycosylation

We have characterized receptors for the insulin-like growth factor (IGF-I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell-free system, IGF-I and insulin stimulated the phosphorylation of 95-kDa and 105-kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF-I receptor beta subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor beta subunit is a separate single 95-kDa protein. Enzymatic digestion of IGF-I receptor beta subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS-PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N-linked complex-type carbohydrate chains on the extracellular domain of beta subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion. Analysis of IGF-I receptor beta subunit subtypes in these cells may provide insights into the mechanism of action of IGF-I on neural tissues.     

ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.     

Changes of soluble proteins in leaf and thylakoid exposed in high saline condition of a mangrove taxa Bruguiera gymnorrhiza

One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1–3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.Key words: Bruguiera gymnorrhiza, Mangrove, Polypeptides, Salt shock, Sodium chloride, Thylakoid     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.