A detailed examination of the iodination of beta-galactosidase: stoichiometric inactivation by nonspecific iodination

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.     

Cell Cycle Phase Sensitivity to Cis-Dichloro-Bis (Isopropylamine) Trans-Dihydroxy Platinum (Iv) (Chip)

Abstract. Cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum (IV) (CHIP) is a second generation platinum coordination complex now in Phase II clinical trials. In vitro studies with Chinese Hamster Ovary cell cultures show that CHIP is a phase-sensitive drug, being most cytotoxic to cells in early G₁ phase and least toxic to late S and G₁ phase cells. the dose-modifying factor between the drug sensitivity of cells treated in G₁ and in late S phase is 1.6. These findings and their clinical significance are discussed with respect to the phase sensitivity of other cytotoxic agents.     

Control of the activation/inactivation of pyruvate, Pi dikinase from the C4 plant maize by adenylate energy charge, pyruvate, and analogs of pyruvate

Pyruvate, Pi dikinase, which is localized in the mesophyll chloroplasts of C4 plants, requires a high adenylate energy charge for conversion of the enzyme from the inactive to the active form. The inactivation process is favored by a low energy charge, being maximal at values below 0.7. Pyruvate and analogs of pyruvate, oxamate and oxalate, strongly inhibit the inactivation process at millimolar levels. The results suggest that light activation of the enzyme in vivo may be mediated by an increased adenylate energy charge in the chloroplast. Pyruvate may allow a higher steady-state level of activation to be achieved in vivo by inhibiting inactivation.     

chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase.

The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.     

Conformational changes in Sindbis virus envelope proteins accompanying exposure to low pH.

The attachment of high multiplicities of Sindbis virus to tissue-cultured cells followed by brief treatment at low pH has been shown to produce cell fusion (fusion from without). In this report, experiments to determine the effects of low pH on the physical and biological properties of Sindbis virus are described. Exposure of purified Sindbis virions to mildly acidic conditions resulted in a rapid and irreversible alteration in particle density and sedimentation characteristics, followed by a slower loss of infectivity. Infectivity was not restored by a return to neutral pH; rather, the loss of virus infectivity seemed to be initiated by exposure to low pH but continued at neutral pH. The formation of a virus-cell complex in which virions were attached to the cell surface protected the particles from low-pH inactivation, although low pH could still expose virus functions responsible for cell fusion. Low pH was found to induce a conformational change in the E2 polypeptide of the intact virion. These results are discussed with respect to the process of Sindbis virus infection of tissue-cultured cells.     

Response of sows to oestradiol benzoate treatment after weaning at two stages of lactation

The timing and dosage of oestradiol benzoate injected after weaning was critical with respect to the pattern of behavioural oestrus and the ovarian stimulation achieved; treatment on the day of weaning (Day 0) and Day 1 with 60 micrograms oestradiol benzoate/kg body wt produced poor ovulatory responses and abnormal oestrous behaviour. Treatment on Day 2 with 30 micrograms oestradiol benzoate/kg resulted in consistent oestrus and ovulatory responses although the ovulation rates (10 . 6 +/- 1 . 1 in 3-week and 12 . 2 +/- 1 . 7 in 5-week weaned sows) were below those expected in fertile untreated sows weaned at these times. The timing of the preovulatory LH surge (53 . 6 +/- 2 h after oestradiol benzoate) was closely synchronized in all sows and a similar synchronous rise in plasma progesterone concentrations 100 +/- 4 h after oestradiol benzoate suggests a similar synchrony of ovulation. The magnitude of the LH and FSH responses to oestradiol benzoate were similar to those that occur in untreated sows and similar differences also existed in gonadotrophin secretion related to the length of lactation.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Inhibition of human lymphocyte activation by wheat germ agglutinin: a model for saccharide-specific suppressor factors

The suppressive effect of wheat germ agglutinin (WGA) on lectin-stimulated blastogenesis and immunoglobulin production was studied. Addition of WGA at 10 micrograms/ml inhibited phytohemagglutinin (PHA)-, concanavalin-A (Con-A)-, and pokeweed mitogen (PWM)-induced mitogenic responses by 70-80%. PWM-driven immunoglobulin synthesis was suppressed by 45% with WGA. The inhibitory effects of WGA were not due to cell death or to interference with lectin binding at the cell surface. Inhibition was dependent on the presence of WGA in the cell culture during the first 24 hr of mitogen exposure and was observed in cultures of both monocyte-depleted peripheral blood mononuclear cells as well as T-cell-enriched populations. WGA-induced inhibition of blastogenesis was blocked by the addition of N-acetylglucosamine (GluNAc) which prevents WGA binding to the cell surface. WGA was found to mimic the suppressive effect of a soluble immune suppressor supernatant (SISS) derived from Con-A-activated mononuclear cell cultures. PHA responses were inhibited by 80 and 95% with SISS and WGA, respectively. The inhibition by both WGA and SISS was totally reversed with addition of GluNAc. Furthermore, WGA and SISS demonstrated competition for the same cell surface receptor site. WGA may therefore be useful as an in vitro model of a saccharide-specific, biologically relevant, soluble mediator for the investigation of mechanisms of immunologic suppression.     

Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.