Cloning and characterization of a fox sperm protein FSA-1

1994 ◽  
Vol 6 (6) ◽  
pp. 761 ◽  
Author(s):  
S Beaton ◽  
A Cleary ◽  
Have J ten ◽  
MP Bradley
Keyword(s):  
Fetal Brain ◽  
Protein Band ◽  
Major Protein ◽  
Structural Protein ◽  
Testicular Germ Cell ◽  
Western Blots ◽  
Sperm Protein ◽  
Major Protein Band ◽  
High Stringency ◽  
Rna Transcript

A monoclonal antibody was raised to a fox sperm protein (FSA-1) which was found to be localized to the inner acrosomal compartment of sperm fixed in methanol. Western blots of testicular germ cell membrane extracts probed with this antibody identified a major protein band with a molecular weight of 36,000. Immunofluorescent studies on fox testis sections showed that the antigen is expressed on round and elongating spermatids on a crescent-shaped structure, which probably represents the developing acrosome. An antibody specific for FSA-1 was used to screen a fox testis cDNA library for its cognate gene. An 875-bp cDNA clone was isolated and sequenced revealing an open reading frame. Searches of the GenBank and EMBL databases with the nucleic acid sequence revealed significant homology (86%) of FSA-1 with 406 bases of an unidentified RNA transcript from human fetal brain (EST02625). Northern blot analysis of fox testis RNA samples identified an RNA transcript of approximately 0.9 kb during the months when spermatogenesis is active. Zoo Northern blots (at high stringency) reveal an RNA transcript of a similar size present in testis RNA from dogs and mice. Zoo Southern analysis (high stringency) reveal genomic sequences present in dogs, mice, cattle and sheep. At present, the function of the FSA-1 gene product remains unknown, but it may play a role as a structural protein component of the acrosome.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig

1991 ◽  
Vol 260 (6) ◽  
pp. G865-G872 ◽  
Author(s):  
C. J. Chandler ◽  
D. A. Harrison ◽  
C. A. Buffington ◽  
N. A. Santiago ◽  
C. H. Halsted
Keyword(s):  
Brush Border ◽  
Protein Band ◽  
Major Protein ◽  
Functional Specificity ◽  
Major Protein Band ◽  
Regional Location ◽  
Minor Protein ◽  
A Minor ◽  
Hydrolysis Of

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

Revised immunolocalization of the Na+-d-glucose cotransporter SGLT1 in rat organs with an improved antibody

2008 ◽  
Vol 295 (2) ◽  
pp. C475-C489 ◽  
Author(s):  
Daniela Balen ◽  
Marija Ljubojević ◽  
Davorka Breljak ◽  
Hrvoje Brzica ◽  
Vilim Z̆lender ◽  
...  
Keyword(s):  
Small Intestine ◽  
Protein Expression ◽  
Rat Kidney ◽  
Protein Band ◽  
Tissue Expression ◽  
Major Protein ◽  
Rat Organs ◽  
Major Protein Band ◽  
Outer Stripe ◽  
Intracellular Organelles

Previously, we characterized localization of Na+-glucose cotransporter SGLT1 ( Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Škarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913–926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of ∼75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.

scholarly journals Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5122-5129 ◽  
Author(s):  
Charles C. Chu ◽  
Rosa Catera ◽  
Katerina Hatzi ◽  
Xiao-Jie Yan ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Band ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Major Protein ◽  
Nonmuscle Myosin ◽  
Cell Extracts ◽  
Major Protein Band

Abstract Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids

1988 ◽  
Vol 118 (3) ◽  
pp. 501-NP ◽  
Author(s):  
S. Mac Neil ◽  
R. A. Dawson ◽  
G. Crocker ◽  
C. H. Barton ◽  
L. Hanford ◽  
...  
Keyword(s):  
Growth Factor ◽  
Breast Milk ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Biologically Active ◽  
Major Protein ◽  
Major Protein Band ◽  
Normal Human ◽  
Epidermal Growth

ABSTRACT In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band co-migrating with authentic calmodulin (Mr 17 000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14 000–15 000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0·03 nmol/l in urine to 18·6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0·79, P < 0·01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products. J. Endocr. (1988) 118, 501–509

Isolation and Characterization of Sepiapterin Reductase from Drosophila melanogaster

Pteridines ◽  
1993 ◽  
Vol 4 (1) ◽  
pp. 43-50 ◽  
Author(s):  
K.H. Yoon ◽  
K.W. Cha ◽  
S.I. Park ◽  
J.J. Yim
Keyword(s):  
Drosophila Melanogaster ◽  
Carbonyl Compounds ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Major Protein ◽  
Sepiapterin Reductase ◽  
Isolation And Characterization ◽  
Major Protein Band

Summary Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH4). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe3+ . Sn4+ and divalent cation. Cd2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds

The effect of ageing cooled milk on the composition of the fat globule membrane

1972 ◽  
Vol 39 (1) ◽  
pp. 95-105 ◽  
Author(s):  
M. Anderson ◽  
G. C. Cheeseman ◽  
Dorothy J. Knight ◽  
W. F. Shipe
Keyword(s):  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Band ◽  
Major Protein ◽  
Fresh Milk ◽  
Major Protein Band ◽  
Fat Globule

SummaryThe effect of ageing fresh milk for 24 h at 4°C on the composition of 4 fractions of milk fat globule membrane (FGM) – microsomal pellet membrane (MPM), deoxycholate soluble membrane (DOCM), high density membrane (HDM) and low density membrane (LDM) – prepared from washed cream treated with sodium deoxycholate (DOC), was studied for milk of individual cows. Total FGM and its fractions were solubilized by treatment with sodium dodecyl sulphate (SDS), EDTA and β-mercaptoethanol, and the dissociated FGM proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS.Ageing resulted in a greater loss of phospholipid during cream preparation than was found with fresh milk, and also in a reduction in the total amount of FGM isolated. Loss of FGM material was confined to MPM, DOCM and LDM and was accompanied by compositional changes in DOCM and LDM, quantitatively the most significant fractions. Ageing also produced changes in the gel electrophoresis patterns of DOCM, where the band of greatest mobility (mol. wt about 16000) was partially lost, and of LDM, where there was an increase observed in the major protein band.The implication of the results on the proposed models of FGM structure is discussed.

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

1989 ◽  
Vol 35 (6) ◽  
pp. 651-655 ◽  
Author(s):  
L. J. Ward ◽  
S. H. De Boer
Keyword(s):  
Monoclonal Antibody ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Protein Band ◽  
Major Protein ◽  
Western Blots ◽  
Diverse Range ◽  
Purified Antigen

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41-and a 44-kilodalton protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulphate – polyaerylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.Key words: Erwinia carotovora, pectate lyase, monoclonal antibody.

scholarly journals Thrombokinase of the Blood as Trypsin-Like Enzyme

1962 ◽  
Vol 45 (4) ◽  
pp. 103-113 ◽  
Author(s):  
J. H. Milstone
Keyword(s):  
Methyl Ester ◽  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Protein Band ◽  
Soybean Trypsin Inhibitor ◽  
Major Protein ◽  
Bovine Plasma ◽  
Major Protein Band

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.

scholarly journals Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913 ◽  
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

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