Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA

We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon. By normalizing for each analogue's previously determined affinity for the trp aporepressor, we have estimated the extent to which each of the functional groups of bound L-tryptophan contributes to operator affinity. We discuss the likely role of these functional groups in the context of the crystal structures of the inactive, unliganded trp aporepressor, the liganded, active repressor, an inactive pseudorepressor (Pseudorepressors are formed by analogues of L-tryptophan that bind at the tryptophan-binding site but form near isomorphs of the repressor that have poor affinity for operator-DNA.) and the trp repressor/operator complex. We find that the alpha-amino group and an unsubstituted amino (-NH-) nitrogen of L-tryptophan's indole ring are essential for operator affinity. The former properly orients the corepressor and the latter interacts directly with the DNA. The alpha-carboxyl group, on the other hand, greatly enhances but is not essential for operator binding. The alpha-carboxylate's role, which is dependent on the corepressor's orientation in the binding pocket, is apparently to position the guanidinium group of Arg-84 for favorable contacts with the operator's sugar-phosphate backbone.     

Otolith microstructure indicating growth and mortality among plaice, Pleuronectes platessa L., post-larval sub-cohorts

Growth and mortality of post-metamorphosed plaice were studied by means of daily increments in the sagittal otoliths. The Gompertz model was the best fit to length-at-age data and there were no significant differences between length-at-age and back-calculated lengths. The microstructure pattern of the otoliths at metamorphosis was also used to estimate hatching and settlement distributions. Differential growth and mortality occurred among sub-cohorts; growth rates and mortality were higher in fish that settled earlier. In 1986, the best survival was for a sub-cohort settling in late May to early June. In contrast, in the warmer season of 1987, survival was highest for the second and third sub-cohorts settling in late April and mid May.     

Increased regional vascular albumin permeation in the rat during anaphylaxis

The changes in vascular albumin permeation induced by systemic anaphylaxis were studied simultaneously in 21 different tissues of the same animal. Before Ag challenge sensitized rats were injected i.v. with 125I-albumin (test tracer), 51Cr-RBC (vascular space marker) and 57Co-EDTA (extravascular space marker). The index of vascular permeation used was the tissue to blood isotope ratio (tbir), which was obtained by dividing the ratio of 125I/51Cr counts in each tissue by the ratio of the same isotopes in the arterial blood sample. After Ag challenge, the increase in the tbir varied considerably among the different tissues. The most pronounced increase was noted in the lymph node (ninefold) followed by the aorta and mesentery (six- to sevenfold) and the various parts of the gastrointestinal tract (four- to sixfold). In the skin less than skeletal muscle less than lung less than liver and eye two- to fourfold increases occurred. Relatively minor increases in albumin permeation (less than twofold) were observed in the brain less than kidney less than heart and less than spleen. The testis was the only organ in which no significant change occurred. For some of the tissues there was also an increase in the tbir for 57Co/51Cr (an index of the extracellular fluid space) suggesting edema formation. The highest increase was noted in the aorta (fourfold). Minor increases occurred in the atrium of the heart, stomach, duodenum, and lymph nodes. There was also a 36% increase in hematocrit. Therefore, systemic anaphylaxis caused extensive extravasation of albumin and hemoconcentration.     

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.     

The MgATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme.

An ATP-like affinity labeling reagent, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), was used to probe the MgATP-binding site of smooth muscle myosin light chain kinase from chicken gizzard (smMLCK) and its calmodulin (CaM) complex. Native smMLCK has an absolute requirement for the binding of the calcium complex of CaM for expression of its catalytic activity. FSBA reacted with smMLCK-CaM and with the CaM-free, inactive enzyme as well. Both reactions were dependent on time and FSBA concentration. Reaction was accompanied by the incorporation of covalently bound [14C]FSBA into smMLCK protein at a molar ratio of approximately 1:1 in each case. p-(Fluorosulfonyl)benzoic acid, an analogue of FSBA lacking the adenosine targeting group, did not react at a significant rate with either form of smMLCK. Reaction of CaM-free and CaM-bound smMLCK with FSBA displayed saturation kinetics. The first-order rate constants for the conversion of the reversible, noncovalent enzyme-FSBA complex to form the irreversibly inhibited, covalently modified enzyme were similar for both smMLCK and smMLCK-CaM, 0.15 and 0.07 min-1, respectively. The concentrations of FSBA yielding the half-maximal rate of inactivation, KI, were essentially identical--0.65 and 0.64 mM, respectively--for smMLCK and smMLCK-CaM. MgATP, but not MgGTP or a substrate peptide, potently inhibited reaction with FSBA. Inhibition by MgATP was competitive. The measured inhibitory constant for MgATP was essentially the same--33 versus 34 microM--for both smMLCK and smMLCK-CaM. It therefore is concluded that the MgATP-binding site on smMLCK remains accessible and recognizable as such when the enzyme becomes inactivated upon dissociation of CaM.     

Sequence-dependent distortions induced in DNA by monofunctional platinum(II) binding.

The effects on thermal stability and conformation of DNA produced by the monofunctional adducts of chlorodiethylenetriamineplatinum(II) chloride ([Pt(dien)Cl]Cl) have been investigated. Oligodeoxyribonucleotide duplexes of varying lengths (9-20 base pairs) and of varying central trinucleotide sequences were prepared and characterized that contained site-specific and unique N(7)-guanine adducts. Included are adducts at the sequences of d(AGC), d(AGT), d(CGA), d(TGA), d(TGC), and d(TGT). All these monofunctional adducts decrease the melting temperature (Tm) of the duplexes. This destabilization effect exhibits a sequence-dependent variability. The highest lowering of Tm is observed for the modified duplexes containing the central sequence of pyrimidine-guanine-pyrimidine. The destabilization effect is reduced with decreasing concentrations of Na+. Polarography, circular dichroism, phenanthroline-copper, and chemical probes reveal conformational distortions spreading over several base pairs around the adduct. The effects of monofunctional platinum(II) adducts on conformational distortions in DNA exhibit a sequence-dependent variability similar to those on thermal stability of DNA. The influence of the monofunctional adduct formed by cis-diamminemonoaquamonochloroplatinum(II) on the stability of the oligonucleotide duplex has been also studied. This lesion decreases thermal stability of DNA in the same way as does the adduct of [Pt(dien)Cl]Cl.