Effect of N,N,N',N'-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels

Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.     

Activity-dependent accumulation of basal lamina by cultured rat myotubes

Myoblasts from 20-day rat embryos fuse and differentiate in culture to form spontaneously active myotubes. The myotubes acquire an extracellular matrix that includes a patchy basal lamina (BL) and a layer of fibrils that runs among and above the cells. Several antibodies that bind to muscle fiber basement membrane in vivo were used to study the organization of the extracellular matrix and the effect of muscle activity on the accumulation of its components. Light and electron microscopic immunohistochemical methods showed that the composition and organization of myotube BL in vitro resemble those seen in vivo. Antibodies that bind to both synaptic and extrasynaptic muscle fiber BL, in vivo stain the entire myotube BL in vitro, while antisera that bind preferentially to synaptic BL in vivo stain small patches of myotube BL, which are usually associated with regions rich in acetylcholine receptors. The effects of activity on accumulation of BL were studied by comparing control myotubes to myotubes paralyzed with tetrodotoxin or lidocaine. Immunohistochemical and 125I-antibody binding experiments with three antisera that stain the entire BL showed that paralyzed myotubes accumulate less BL than active myotubes. The effects of activity and inactivity are reversible: new BL forms if toxin is removed from cultures and BL is lost if active myotubes are paralyzed. Thus, accumulation of BL by myotubes is dependent, at least in part, on activity. In contrast, the number of patches stained by synapse-specific BL antibodies is increased in inactive cultures. Thus, immunologically distinguishable components of BL are differentially affected by activity.     

Augmentation of IgE-mediated release of histamine by 5-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid

Highly purified rat mast cells converted 1.12% and 1.64% of exogenously added [1-¹⁴C]arachidonic acid to 5-OH-6,8,11,14 eicosatetraenoic acid (5-HETE) and 12-OH-5,8,10,14 eicosatetraenoic acid (12-HETE) respectively during a three minute incubation at 37°. Both 5-HETE and 12-HETE (1–10 μM) augmented the histamine release response to goat anti-rat IgE antibody (a reverse anaphylaxis system). These results indicate that mast cells synthesize 5-HETE and 12-HETE and that these molecules can enhance mediator release.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

Increased serum prolactin levels mediate the suppressive effects of ectopic pituitary grafts on copulatory behavior in male rats

To determine if deficits in sexual activity observed in pituitary-grafted male rats are due to elevated serum prolactin (PRL) levels found in these animals, the effects of whole pituitary grafts, pars distalis grafts, and ovine (o) PRL treatment on male copulatory behavior were compared. Adult sexually experienced CDF male rats were given four whole pituitary grafts, four pars distalis grafts, or were sham operated. Both groups of grafted animals exhibited suppressed copulatory behavior patterns when tested 18 days after pituitary transplantation. Animals given whole pituitary grafts had significantly longer latencies to mount (P less than 0.05) and to intromit (P less than 0.01) than did the sham-operated controls, while the animals given anterior pituitary grafts differed from the sham-operated controls in latencies to mount (P less than 0.05) and to intromit (P less than 0.01), as well as in the number of intromissions (P less than 0.05). Prolactin-injected animals had significantly reduced intromission rates (P less than 0.01) and significantly increased latencies to mount (P less than 0.05) and to intromit (P less than 0.01) when compared to vehicle-injected controls. Furthermore, the time course of behavioral suppression was similar in oPRL-treated animals to that observed in pars distalis-grafted males, with both groups showing the onset of deficits in sexual activity within 8 to 9 days from the induction of the hyperprolactinemic state. The similarity in pattern and time to onset of behavioral suppression in pituitary-grafted and oPRL-treated animals suggests that behavioral deficits observed in animals with pituitary grafts result from chronic elevation of serum PRL levels.     

Automatic collector of expired 14CO2 for liquid scintillation counting

A filtration technique was employed to trap ¹⁴CO₂ continuously for liquid scintillation counting. Devices for delivering scintillator and ethanolamine solutions were combined symmetrically with two fritted-glass aspirators for altenating operation. The collector was regulated by a fraction collector timer. Trial and animal tests indicated that the described method was efficient, reliable, and more convenient for frequent collection over long periods than alternative methods. The automatic collector was used for metabolic studies of [1-¹⁴C] arachidonic acid in rats kept in metabolic cages and the results were processed by multicompartmental analysis.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Identification of a protein from escherichia coli whose synthesis appears to be triggered by the initiation of DNA replication

Using a DNA temperature sensitive initiation mutant to synchronize the replication and cell division cycle, we have compared proteins which are synthesized during a period of DNA arrest with those synthesized after return to permissive temperature. This work has led to the identification of a DNA-binding protein of 60–65,000 molecular weight (SDS-gel electrophoresis) whose synthesis appears to be triggered by the initiation event.     

Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

The mechanism of cell-mediated cytotoxicity. IV. K-76 COONa, which inhibits the activity of Factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

Identification of several species of phospholipase inhibitory protein(s) by radioimmunoassay for lipomodulin

Radioimmunoassay of lipomodulin has been developed using a monoclonal anti-lipomodulin antibody and ¹²⁵I-labelled lipomodulin. Lipomodulin activity was measured in peritoneal lavage fluids obtained from rats injected with dexamethasone by radioimmunoassay and by enzymatic assay with phospholipase A₂. Three species of immunoreactive substances with Mr= 40,000, 30,000 and 16,000 were found. While two species of Mr= 40,000 and 30,000 had phospholipase inhibitory activities, the species of Mr= 16,000 could inhibit phospholipase A₂ only after dephosphorylation by alkaline phosphatase treatment.     

Identification of a peptide sequence involved in association of subunits of yeast hexokinases

The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.     

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of $\text{Rhus}$ laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do $\text{not}$ appear to bridge the type 3 binuclear copper ions in laccase.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Effects of catecholamines on ammoniagenesis and gluconeogenesis by renal cortex in vitro

Norepinephrine (arterenol) and a synthetic catecholamine, isoproterenol, increase the production of ammonia and glucose from glutamine and glutamate by rat renal cortical slices in vitro. The stimulation of both ammonia and glucose production by isoproterenol was greater than that observed with identical molar concentrations of arterenol. Isoproterenol markedly increased the concentration of cyclic AMP in rat renal cortical slices. Addition of propranolol, a β-adrenergic blocking agent, prevented the increase of cyclic AMP levels induced by isoproterenol. Cyclic AMP increased both ammoniagenesis and gluconeogenesis by kidney cortex. Thehe increase in ammonia production produced by isoprotenol was blocked by the addition of propranolol. It is concluded that the increase in ammonia and glucose production caused by isoproterenol is mediated through the release of cyclic AMP.