Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.

Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.     

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

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.     

A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures

A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance.     

Modulation of aortic protein phosphorylation by benzo(a)pyrene: Implications in PAH-induced atherogenesis

The toxicity of polycyclic aromatic hydrocarbons such as benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and 3-methylcholanthrene has been associated with alterations in the proliferation of vascular smooth muscle cells and the development of lesions of mesenchymal origin. Because phosphorylation of endogenous substrates plays a central role in the regulation of smooth muscle cell growth, the present studies were conducted to evaluate the phosphorylation pattern of medial aortic protein upon repeated in vivo exposure of Japanese quail to benzo(a)pyrene (BaP). Medial aortic homogenates from quail treated for 10 weeks with 10 mg/kg benzo(a)pyrene or vehicle were processed for in vitro measurements of protein phosphorylation. In vitro phosphorylation of endogenous or exogenous proteins stimulated in vitro by phorbol myristate acetate/phosphatidyl-serine or cyclic AMP, known activators of protein kinase C and cyclic AMP-dependent protein kinase, respectively, was examined in the cytosolic and particulate fractions of homogenates from control and treated animals. Benzo(a)pyrene treatment significantly enhanced the basal phosphorylation of Mr 113, 35, and 23 kDa proteins in the cytosolic fraction. Modest increases in the phosphorylation of Mr 71, 52, and 38 kDa were also observed under basal conditions. No changes in the basal phosphorylation of particulate proteins were observed. Phosphorylation of endogenous protein substrates by protein kinase C in the cytosolic fraction was not altered by benzo(a)pyrene treatment. In contrast, inhibition of C-kinase-mediated phosphorylation of endogenous Mr 272, 72, and 45 kDa proteins was observed in the particulate fraction of aortic homogenates from benzo(a)pyrene-treated quail relative to controls. Exogenous histone phosphorylation by PKC in the particulate, but not cytosolic fraction, was decreased by benzo(a)pyrene treatment. The effects of benzo(a)pyrene on the C-kinase system were specific, since cAMP-mediated phosphorylation of endogenous proteins, as well as exogenous histone, was not altered by benzo(a)pyrene. Interestingly, benzo(a)pyrene treatment was associated with a selective increase of Mr 200, 80, and 67 kDa proteins in the cytosolic fraction. Collectively, these data are consistent with the hypothesis that medial protein phosphorylation is a significant molecular target of benzo(a)pyrene within the vascular wall.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Endothelin-1 effect on tyrosine phosphorylation and on tyrosine phosphatase (PTP-1C) translocation in rabbit platelets.

This study examined the temporal relationships of endothelin-1-stimulated rabbit platelets tyrosine phosphorylated proteins. The effect of endothelin-1 on tyrosine phosphorylation was dose- and time-dependent and caused a rapid tyrosine phosphorylation of three groups of proteins in the molecular mass range 70-100 kDa, 100-150 kDa and 150-200 kDa. Significant protein tyrosine phosphatase activity and amount were found to be associated with the cytoskeleton of endothelin-1-stimulated rabbit platelets. Under our experimental conditions, translocation from the cytosolic fraction to the cytoskeleton reached its highest levels within 10-20 sec of endothelin-1 stimulation. Endothelin-1-induced translocation of protein tyrosine phosphatase, associated with the increase in its activity was demonstrated by immunoblotting and immunoelectron microscopy.     

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.     

Protein phosphorylation associated with stimulation of rabbit gastric glands

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.     

Action of aldosterone on protein expression in cultured collecting duct cells from neonatal rabbit kidney

To investigate whether 'aldosterone-induced proteins' could be detected in mammalian species, cultured renal collecting duct epithelia from neonatal rabbit kidneys were labelled under aldosterone administration with radioactive methionine and subsequently fractionated into cytosolic and coarse membrane protein fractions. Newly synthesized proteins were then analyzed by SDS-PAGE, isoelectric focussing and two-dimensional electrophoresis. Quantitative estimates of individual newly synthesized proteins were performed utilizing gel slicing, scintillation counting and autoradiography. The labelling experiments demonstrated that, in comparison to controls, aldosterone (1 X 10(-6) M) generally increased the amount of radioactive protein. No qualitative changes in the pattern of newly synthesized proteins and, therefore, no classical aldosterone-induced proteins were observed. The increase of radioactive protein was already seen after 1, 6, and 18 h of hormone treatment. The effect could be blocked partially by spironolactone (1.5 X 10(-4) M), and totally by amiloride (1 X 10(-6) M), g-strophantin (5 X 10(-4) M), and cycloheximide (1 X 10(-6) M. Thus, the interference of aldosterone action at the receptor level, the Na+ channels and the Na+/K(+)-ATPase pump demonstrate that the expression of proteins in cultured renal collecting duct cells is a sensitive system and seems to be controlled by aldosterone at the receptor level, but also counter-controlled by specific plasma membrane sites.     

Molecular weight of major component proteins in crude saline extract of adult Paragonimus westermani

When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 protein (known as egg protein) was 440 kDa. And MW of other band proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17 kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6(phi) X 70 cm sized Sephacryl S-300 Superfine column and revisualized by Disc-PAGE in 8% gel, the sequence of eluted proteins was band 1, band 2, band 6, band 7 and bands 3, 4, 5 and 8. This elution profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot.     

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.     

The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins.

Ras-related small GTP-binding proteins (SMGs) exist in a cytosolic and a membrane-bound pool. The mechanism regulating the intracellular distribution of SMGs remains to be elucidated. We have, therefore, investigated the properties of SMGs expressed in cells of the insulin-secreting lines RINm5F and HIT-T15. Phase-partitioning analysis revealed that smg25A/rab3A as well as all the SMGs in the 23-27 kDa range, labeled by radioactive GTP after blotting, were hydrophobic, regardless of their subcellular distribution. In contrast, the cytosolic forms of ADP ribosylation factor, rho, and CDC42 were hydrophilic. The cytosolic pool of the 23-27-kDa group, including smg25A/rab3A, sedimented in a sucrose density gradient as complexes with an apparent M(r) of about 80,000, whereas rho and CDC42 were recovered in 45-kDa complexes. ARF, however, was uncomplexed (M(r) close to 20,000). The 80-kDa aggregates were likely to be formed by 1:1 complexes with the regulatory protein smg25/GDP dissociation inhibitor (smg25/GDI). In fact, pure smg25/GDI by sucrose gradient exhibited a molecular mass of 55 kDa, but cosedimented with the 80-kDa complexes in cytosolic extracts of insulin-secreting cells. Moreover, purified smg25/GDI was able to extract the SMGs of the 23-27-kDa group from the membranes. Similarly, in cytosolic extracts, rho/GDI cosedimented with the 45-kDa aggregates. Blocking the synthesis of isoprenoid groups with lovastatin resulted in the appearance in the cytosol of SMGs that were hydrophilic. These SMGs were found to sediment with an apparent M(r) close to 25,000 and to be unable to form complexes with smg25/GDI. Lovastatin treatment also caused the accumulation of the noncomplexed form of CDC42 but not of rho proteins. We propose that 1) except for ARF, all the SMGs detected in the cytosol of insulin-secreting cells are associated in 1:1 complexes with their regulatory proteins; 2) the different SMGs can be subdivided into functional groups according to the regulatory protein bound to them; 3) the formation of the 80-kDa complexes with smg25/GDI and of the CDC42 complexes with rho/GDI necessitate the correct carboxyl-terminal post-translational modification of the SMGs.     

Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Differential nuclear localization of the cancer/testis-associated protein, SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

Tyrosine residues of the extrinsic 23 kDa protein are important for its interaction with spinach PSII membranes

The 1, 4, and 8 tyrosine (Tyr) residues on the PSII extrinsic 23 kDa protein were modified with 5, 10 or 40 mM N-acetylimidazole (NAI) respectively. The amount of rebound NAI-modified extrinsic 23 kDa protein was 98%, 80%, and 5% of that in the unmodified protein, respectively. These results indicate that the Tyr residues are absolutely essential to reconstitution ability. Further, the fluorescence and circular dichroism spectra among native and NAI-modified extrinsic 23 kDa proteins were similar, suggesting that the modification by NAI did not markedly influence the basic secondary structure of the native conformation. Thus, we have concluded that the tyrosine residues in the extrinsic 23 kDa protein are important for interaction with PSII membranes. In addition, we found that the structure of the extrinsic 23 kDa protein is stable in suspension (pH 4-9 or Tm 25-55 degrees C).     

Function of the 23 kDa extrinsic protein of Photosystem II as a manganese binding protein and its role in photoactivation

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.     

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.     

Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Isolation and characterization of a 23 kDa protein essential for photosynthetic oxygen evolution

A 23 kDa protein has recently been demonstrated to participate in photosynthetic oxygen evolution by reconstitution experiments on inside-out thylakoid vesicles (Åkerlund H-E, Jansson C and Andersson B (1982) Biochim Biophys Acta 681:1–10). Here we describe the isolation of the 23 kDa protein from a spinach chloroplast extract using ion-exchange chromatography. The protein was obtained in a yield of 25% and with less than 1% of contaminating proteins. The ability of the protein to stimulate oxygen evolution in inside-out thylakoids was preserved throughout the various fractionation steps. The isolated protein was highly water soluble and appeared as a monomer. Its isoelectric point was at pH=7.3. The amino acid composition showed a high content of polar amino acids, resulting in a polarity index of 49%. The isolated protein lacked metals and other prosthetic groups. Its function as a catalytic or regulating subunit in the oxygen evolving complex is discussed.Abbreviations kDa kiloDalton - PAGE polyacrylamide gel electrophoresis