Dalton Species Research Articles

The stability of 99Tcm directly labelled to an Fab' antibody via stannous ion and mercaptoethanol reduction.

The anti-CEA FO23C5 F(ab')2 antibody was directly radiolabelled with 99mTcm by two methods (stannous ion and mercaptoethanol reduction) and compared in vitro and in vivo for label stability. By both methods, reduction of the F(ab')2 fragment produced primarily Fab' fragments. By both methods, the label was stable to 99Tcm-pertechnetate formation in vitro. Analysis by high performance liquid chromatography (HPLC) of serum, urine, kidney and liver homogenates from mice injected with 99Tcm-antibodies by both methods consistently showed a prominent radiolabelled peak with an estimated molecular weight of about 300 daltons. An identical peak was observed in the analysis of patient samples in a related investigation from this laboratory. Cysteine was radiolabelled with reduced 99Tcm and analysed by HPLC and thin layer chromatography (TLC); one of the 99Tcm-cysteine species so produced showed the same chromatographic behaviour as that of the 300 dalton species. In conclusion, the FO23C5 and other antibodies are stably labelled with 99Tcm via either stannous ion or mercaptoethanol reduction. In mice and in patients, the labelled proteins are either catabolized or, more likely, the 99Tcm label is transchelated such that the label is present on several low molecular weight species, the most prominent of which is postulated to be 99Tcm-cysteine.

Purification and Characterization of Lysine-Sensitive Aspartate Kinase from Maize Cell Cultures

Aspartate kinase is a feedback-regulated enzyme that controls the first step common to the biosynthesis of lysine, threonine, isoleucine, and methionine in plants. Aspartate kinase was purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures for physical and kinetic characterization studies. Partial purification and elution from an anion exchange column resolved two lysine-sensitive aspartate kinase isoforms. Both isoforms were purified >1,200-fold to a minimum specific activity of 18 units/milligram of protein. Both isoforms were sensitive to the lysine analogues S-2-aminoethyl-l-cysteine, l-lysine ethyl ester, and delta-hydroxylysine. No threonine-sensitive form of aspartate kinase was detected at any stage during the purification. Additional purification steps were combined with preparative gel electrophoresis to obtain apparently homogeneous lysine-sensitive aspartate kinase. Aspartate kinase appeared to be a tetramer with a holoenzyme molecular weight of 254,000 and to be composed of 49,000 and 60,000 subunits. The tetramer appeared to disassociate during native gel electrophoresis to 113,000 dalton species that retained aspartate kinase activity.

The murine lymphocyte receptor for IgE. V. Biosynthesis, transport, and maturation of the B cell Fc epsilon receptor.

The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.

Pharmacokinetic studies of highly purified human prolactin in normal human subjects.

Previous estimates of PRL pharmacokinetics have been made using radioiodinated human PRL (hPRL) infusions or by measuring serum PRL disappearance following prolactinoma resection. The recent purification of hPRL in significant quantities made it possible to measure the clearance and volume of distribution directly. Studies were also carried out to determine absorption and clearance after im injection. In five normal men whose endogenous PRL secretion was suppressed by dopamine, a loading dose of hPRL (70-90 micrograms) followed by a constant infusion (1.39-2.9 ng/min) produced steady state serum PRL levels of 15.2-25.4 ng/mL by 30-60 min. The calculated mean volume of distribution was 7.3 +/- 2.9 (+/- SD) L. The calculated MCR was 71 +/- 19 mL/min X m2, and the calculated production rate was 802 +/- 377 micrograms/24 h X m2. The plasma disappearance half-life following discontinuation of the infusion was 37 +/- 10 min. The PRL infusate consisted primarily (75.6%) of a 22.5 K dalton species, probably PRL monomer, a component eluting at 45K daltons (16.1% of the total radioimmunoreactivity), probably dimer, and a small amount of a larger mol wt species. Serum obtained during dopamine infusion but before hPRL infusion contained 68.1% of the 22.5K, 7.2% of the 45K, and 24.7% of the larger mol wt moieties. During hPRL infusion in two men there was a relative decrease in the proportion of PRL monomer to 55% and 69% and a relative increase in the PRL dimer to 33% and 18%, respectively. hPRL was injected im in doses of 1, 2, 4, and 8 micrograms/kg without prior dopamine infusion. No significant changes in serum PRL levels occurred after the 1 and 2 micrograms/kg doses (n = 5). After the 4 micrograms/kg dose (n = 8), mean serum PRL levels rose from 10.0 +/- 1.8 (+/- SEM) ng/mL to peak levels of 13.1 +/- 1.8 ng/mL (P less than 0.01). After the 8 micrograms/kg dose (n = 7), PRL levels rose from 9.3 +/- 1.6 to 16.5 +/- 1.8 ng/mL (P less than 0.01). The PRL rise began between 60 and 80 min after injection; peak levels occurred at 160-180 min. In two men given 8 micrograms/kg who were sampled for an additional 3 h, PRL levels peaked at 200-220 min and began to fall by 220-240 min, but had not returned to baseline by 6 h. There were no side-effects of PRL administration, although the 8 micrograms/kg dose caused transient local discomfort.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of plasminogen activator and plasminogen activator-inhibitor production by tissue culture cells: Evidence for independent induction and regulation

Human bladder carcinoma cells (T-24) were found to secrete plasminogen activator activity when cultured under serum-free conditions. The activity co-migrated with high molecular weight human urokinase when analysed by SDS-PAGE followed by zymography and it could be inhibited by anti-human urokinase. Treatment of the cells with phorbol myristate acetate led to enhanced secretion of the urokinase-like enzyme as well as induced the appearance of an 85 000 dalton plasminogen activator in the conditioned medium. The 85 000 dalton species of activity could also be inhibited by anti-human urokinase antibodies and was shown to consist of an SDS-stable complex between the 52 000 dalton high molecular weight urokinase-like plasminogen activator and a 33 000 dalton inhibitor. The urokinase-like enzyme was always produced in excess compared with the 33 000 dalton inhibitor. Phorbol myristate acetate induced stimulation of plasminogen activator synthesis occurred rapidly (1 h) while the appearance of the 85 000 enzyme-inhibitor complex was not evident until 5 h after addition of the phorbol myristate acetate. Addition of several compounds (LiCl, dexamethasone, gamma-interferon) to cultures of T-24 cells lead to a depression of plasminogen activator secretion. In contrast, addition of cyclic AMP to the cultures did not inhibit enzyme production but completely suppressed complex formation. Dexamethasone inhibited both PA secretion and inhibitor secretion while LiCl and gamma interferon only inhibited enzyme secretion. Thus, some biologically active molecules can regulate the production of the two components co-ordinately or independently.

Monoclonal Antibodies to Human Lysyl Oxidase

Hybridoma antibodies against human lysyl oxidase were produced by fusing Sp. 2.0-Ag 14 myeloma cells with spleen cells from mice hyperimmunized with lysyl oxidase isolated from umbilical cords. Hybridomas positive by enzyme-linked immunosorbent assay (ELISA) for human lysyl oxidase were cloned by the dilution method. Eight hybridomas producing antibodies were isolated, three of which also recognized purified bovine aortic lysyl oxidase in an ELISA. One of these antibodies, monoclonal antibody I, was purified and attached to Sepharose CL-4B. The immobilized antibody was effective in binding an enzymatically active 30,000 dalton species. Immunoblot analysis of four of these antibodies showed reactivity against dic 30,000 dalton catalytically active enzyme and the 24,000 dalton fragment of lysyl oxidase. These monoclonal antibodies should be useful tools for studying the localization and biosynthesis of lysyl oxidase.

Distinct hydrodynamic forms of the insulin receptor: electrophoretic analysis of the RI and RII species.

In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do represent larger (RI) and smaller (RII) oligomeric forms of the receptor, rather than representing receptor species separated from each other by differential adsorption to the Sepharose matrix. Receptor solubilized from isolated human placenta membranes was purified by lectin- and insulin-agarose chromatography and was radiolabeled with carrier-free 125I. The labeled receptor was separated by Sepharose 6B gel filtration into two fractions (peak I, KAV = 0.31; peak II, KAV = 0.53), was immunoprecipitated by anti-insulin receptor antibody, and was analysed by electrophoresis in nonreducing polyacrylamide slab gels. The autoradiograms of the gels indicated that peak I (KAV = 0.31, RI receptor form) contained a number of receptor species of 240 000 daltons or greater, whereas peak II (KAV = 0.53, RII receptor form) contained mainly receptor species of 210 000 daltons or smaller. In particular, large amounts of a 90 000 dalton species (presumably free receptor beta-subunit) were present in peak II. Incubation of the material obtained from peak I with insulin resulted in a change in the electrophoretic pattern, which became identical with that observed for material recovered from peak II.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the polymorphonuclear leukocyte C5a receptor.

The peptide C5a is thought to play an important role in the inflammatory response primarily through its action on the polymorphonuclear leukocyte (PMN). The receptor for C5a on human PMN has now been identified by affinity labeling. Cross-linking 125I-C5a to intact PMN with disuccinimidyl suberate produced a species that had a molecular mass on sodium dodecyl sulfate gels of 5.2 X 10(4) daltons. We believe this species represents a complex between C5a and its receptor for the following reasons. The band is eliminated if the cross-linking experiment is performed in the presence of a large excess of unlabeled C5a, but is unaffected by the presence of nonspecific protein or the chemotactic factors N-formyl-Met-Leu-Phe and leukotriene B4. The 5.2 X 10(4)-dalton species is not observed if the cross-linker is omitted. Finally, the dose-response curves for the inhibition of binding of 125I-C5a by unlabeled C5a and the inhibition of cross-linking are similar. Subtraction of the molecular mass of C5a from that of the complex gives a molecular mass for the binding moiety of the C5a receptor of 4.0 X 10(4) daltons.

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO(2), but its synthesis was induced under low concentrations of CO(2) (air levels of CO(2)). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO(2) to growth on air levels of CO(2), (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens.

Abstract The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.

A simple method to characterize γ-crystallin synthesized in vitro

Investigation of the migration of in vitro synthesized γ-crystallin which has not been heat denatured on polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate indicates that this class of proteins behaves in an anomalous manner. While other in vitro synthesized lens proteins under these conditions migrate as theoretically expected, γ-crystallin is retarded, having a mobility comparable to a 92 000 dalton component. Only upon heat denaturation does this protein fraction migrate as a 20 000 dalton species. Since other in vitro synthesized lens proteins have molecular weights of approximately 20 000 daltons, it is only with this methodology that γ-crystallin synthesis can be unequivocally followed in a simple, rapid, one-dimensional fractionation system.

High molecular mass phosphoproteins in the rat liver nuclear matrix identification of a prominent 110,000 dalton species

Purified rat liver nuclei were labelled in vitro in the presence of (32P) ATP and submitted to sequential extraction with DNAse, 0.4 or 2.0 M NaCl and Triton X-100. The residual or matrix structures contained 8–10 phosphoproteins between 76 and 260 kd including a triplet of major bands with 110, 117 and 128 kd. The 110 kd species was purified by chromatography on oligo(dT)-cellulose. It was shown to be identical with the 110 kd phosphoprotein of rat liver or Morris hepatoma free polyribosomes using the technique of limited digestion with S. aureus protease V 8.

Functional identification of serum complement components following electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate

A method is described for detecting the active complement components C6 and C7 after polyacrylamide gel electrophoresis (PAGE) of whole serum in the presence of sodium dodecyl sulphate (SDS). The method involves the removal of SDS by washing with non-ionic detergent followed by the application of an erythrocyte/agarose gel to detect haemolytic activity. Two forms of human C6 with apparent molecular weights of approximately 121,000 daltons and 114,000 daltons were observed. Major activity resided in the 121,000 dalton species. The 2 forms of human C6 were not related to known genetic polymorphisms for this component. Analysis of sera from different animal species showed that not all possessed the 2 forms of C6 and that there were interspecies differences in C6 molecular weights. These are most marked in the case of human and murine C6; the major form of murine C6 had a molecular weight approximately 20,000 daltons less than the major human form. One form of human C7 with an apparent molecular weight of 104,000 daltons was seen. The molecular weights of C7 from the various animal sera tested did not differ significantly from this. Studies with reducing agents and metabolic inhibitors showed that both C6 and C7 required intact disulphide bonds and sulphydral groups for functional activity.

Amplification of ribonuclease II (rnb) activity in Escherichia coli K-12.

A 7.1 kb HindIII-XhoI fragment of E. coli DNA which contains the structural gene for ribonuclease II (rnb) has been cloned in the recombinant plasmid pDK24. At least two constitutively expressed genes are encoded on the fragment as shown by maxicell analysis. On denaturing polyacrylamide gels RNase II appears as a single 72,000 dalton species. The approximate site of transcription initiation of the rnb gene has been mapped. Although derivatives of E. coli harboring pDK24 contained 10-fold more RNase II activity that wild type strains without the plasmid, the degradation rate of mRNA was similar in all strains tested. Strains deficient in both RNase II and polynucleotide phosphorylase appear inviable.

Synthesis of Oat Globulin Precursors

The oat (Avena sativa L.) seed globulin was found to be synthesized in vitro as 60,000 to 64,000 dalton precursors. In vivo protein labeling yielded polypeptides of 58,000 to 62,000 daltons, suggesting cleavage of signal sequences from the precursors. Further cleavage is apparently required to separate the alpha and beta polypeptide sequences which are known to form disulfide-linked 53,000 to 58,000 dalton species in the (alphabeta)(6) holoprotein. The data are discussed with respect to analogous synthesis and processing of some legume 11S storage proteins.

Hydrodynamic and Rejection Properties of a Coil Dialyzer

Compliance of coil dialyzers follows a linear dependence on pressure when measured in saline; compliance measurements with non-wetting fluids are inappropriate and underestimate the blood compartment expansion experienced with pressure. The arithmetic mean of inlet and outlet pressures can be used to predict ultrafiltration rate independent of venous outlet pressure, and blood compartment volume varies both with inlet pressure and blood flow. The rejection of carbohydrate marker solutes increases from about 10% for 1,200 Daltons to 100% for 12,000 Dalton species.

Purification of terminal deoxynucleotidyl transferase from pig thymus: identification of 42,000 and 57, 000 dalton species.

Terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been purified 4365-fold from pig thymus. It was further separated into two molecular forms of 57,000 and 45,000 dalton by Sephadex G-100 gel-filtration. The former sedimented at 4.2s through a sucrose gradient, while the latter, at 3.6s. By a SDS-polyacrylamide gel-electrophoresis, their molecular weights were estimated as 57,000 and 42,000 dalton, respectively. Thus each of the large and small pig TdT consists of a single polypeptide of 57,000 or 42,000 dalton and has no subunit structure. These two forms were indistinguishable in antigenicity by a neutralization assay of 42,000 dalton-TdT antibody. The enzymological properties of 42,000 dalton-TdT from pig thymus were very similar to those of calf TdT which has a two-subunits structure.

Immunoreactive dynorphin in the rat adenohypophysis consists exclusively of 6000 dalton species

Immunoreactive dynorphin in the rat adenohypophysis exhibited an apparent molecular size of approximately 6 kilodaltons (6 kDal) upon characterization by gelfiltration. Essentially no dynorphin-related peptides with a molecular size of dynorphin-(1–17) or dynorphin-(1–8), which constitute the great majority of ir-dynorphin in the rat neurointermediate pituitary and brain, could be detected in the adenohypophysis. The possible presence of putative aggregations of smaller peptides were largely excluded by rechromatography under denaturating conditions in 4 M guanidine-HCL. SDS-gelelectrophoresis revealed that 6 kDal dynorphin consisted of at least three components of similar molecular size. From the predominant form of 6 kDal dynorphin, leucine-enkephalin could be liberated by sequential enzymatic cleavage with trypsin and carboxypeptidase B.

Correlation between a specific molecular weight form of plasminogen activator and metabolic activity of 3T3 cells.

In quiescent cultures of 3T3 cells, plasminogen activator (PA) is found predominantly as a 75,000 dalton species. When quiescent cells are exposed to mitogenic agents such as phorbol myristate acetate, Ca++, or 25% serum, the absolute levels of PA in cell lysates may either increase or decrease. However, a consistent observation is that in the stimulated cultures PA is found predominantly as a 49,000 dalton species. This also is the predominant form of PA in growing and transformed cells. Concomitant with the mitogen-induced stimulation of the 49,000 dalton PA in quiescent cultures is a change in morphology to one that is characteristic of growing and transformed cells. The data suggest that PA is not operative in causing the morphological change that occurs with activation; however, the 49,000 dalton PA in particular is closely related to the pleiotypic response accompanying growth stimulation and transformation.

GTPase from rod outer segments: Characterization by photoaffinity labeling and tryptic peptide mapping

The photoaffinity label [γ- 32P]8-N 3GTP has been used to identify GTP-binding components in highly purified preparations of GTPase from bovine rod outer segments. These preparations contain two major polypeptides of 37,000 and 39,000 daltons. In the presence of photolyzing radiation, [γ- 32P]8-N 3GTP is covalently attached to the 37,000 dalton polypeptide. Tryptic peptide mapping of this polypeptide indicates that it is highly related to the 39,000 dalton species that has been previously identified as a GTP-binding component.