Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

Direct scanning active enzyme gel chromatography with halted flow for detecting molecular weight heterogeneity of active enzymes

As an extension of the method of scanning active enzyme chromatography we have introduced the option of halting flow while the enzyme is still present on the column. Once the flow has been halted, continuous monitoring of the absorbancy profile provides information on the relative distribution of the enzyme activity. When the baseline is relatively stable, one can detect very low levels of enzyme activity and thus monitor for the presence of small amounts of active enzyme of molecular weight different from that of the predominant form. This gives a significant improvement over regular active enzyme chromatography in the qualitative detection of molecular weight heterogeneity, by in effect reducing the noise level of the experiment. The method has been applied to several systems including aldolase from rabbit muscle, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase of bovine heart and hexokinase and glucose-6-phosphate dehydrogenase from yeast.     

Homologies in the active site regions of lactate dehydrogenases

Peptides isolated from several lactate dehydrogenases (EC 1.1.1.27) have been characterized and sequenced. These peptides include much of the substrate binding site as well as the loop of polypeptide chain which shows major conformational changes following coenzyme binding. Despite significant differences in catalytic properties, the amino acid sequence in these two active site regions of the molecule is highly conserved in most cases. A noteable exception is cysteine 165 which at one time was thought to be essential for enzymatic activity. The lactate dehydrogenases investigated were isolated from rabbit muscle, chicken heart, beef heart, and lobster tail.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.     

1,25-Dihydroxyvitamin D3 receptors in a subset of mitotically active lymphocytes from rat thymus

Lymphocytes from the rat thymus gland were separated on Percoll gradient in two subsets. The first subset was enriched in large mitotically active cells and the second in small mitotically inert cells. The former cell preparation possessed a 3.3 S macromolecule with high affinity for 1,25-Dihydroxyvitamin D3 (Kd = 3.3 X 10(-10)M) which bound to DNA cellulose and was eluted from this resin with 0.26 M KCl. In contrast, the small-cell-enriched subset of thymic cells was negative for 1,25-Dihydroxyvitamin D3 specific binding. These findings support evidence from studies in human lymphocytes that there exists an association between mitotic activity and 1,25-Dihydroxyvitamin D3 receptor expression in this class of leukocytes.     

Ternary solvents to investigate proteins at zub-zero temperatures.

Mixtures of water, ethylene glycol and methanol in different volume ratios have been selected to carry out kinetics of enzyme reactions at sub-zero temperatures with the intention to reduce maximally the viscosity. Density, viscosity and dielectric constant values of these mixtures as a function of temperature are reported, as well as values of the protonic activity of several buffers under such conditions. A procedure to avoid or delay the eventual damaging effect of methanol on proteins is described.     

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N-bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu-

-Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.     

Molecular conformation of erabutoxin b; atomic coordinates at 2.5 A resolution.

Atomic coordinates determined from a 2.5 Å electron density map are given for erabutoxin b, a sea snake venom postsynaptic neurotoxin. The principal structural features, anti-parallel β pleated sheet, β bulge and β bends are described. The erabutoxin b structure is discussed as structural prototype of this class of homologous curare-mimetic neurotoxins from both land and sea snakes.     

Biosynthesis of polymyxin by Bacillus polymyxa. I. The status of the biosynthetic multienzyme complex during active antibiotic synthesis and sporulation

The involvement of membrane fractions of Bacillus polymyxa in the early stages of the biosynthesis of the peptide antibiotic polymyxin, was established by (a) incorporation of the precursor amino acid in an acid precipitable form in the absence of protein synthesis, (b) presence of all the component amino acid-activating enzymes, and (c) association of bioassayable polymyxin, in the purified membrane fraction. Polymyxin negative mutants that were also blocked at stage 0 of sporulation were shown to be defective in one or more of their membrane-bound amino acid-activating enzymes. A strong correlation between sporulation and antibiotic production had been indicated by the isolation of these mutants which are revertible simultaneously to Ab⁺ and Spo⁺ traits. During the onset of the rapid phase of the elaboration of polymyxin, a delocalization of one of the membrane-bound enzymes, 2,4-diaminobutyric acid-activating enzyme, to the soluble fraction was observed. Concomitant with this change, the levels of the intracellular protein-bound and free polymyxin was increased in the soluble fraction.     

A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH

A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-[N-morpholine]propanesulfonic acid), the leading ion K⁺ and the trailing ion histidine. The system offers improved resolution of cationic proteins.     

Analysis of visual system development in Drosophila melanogaster: mutations at the Glued locus

We have analyzed several aspects of the development of flies carrying mutations at the Glued locus. Optic lobe abnormalities in individuals heterozygous for the original Glued allele were previously shown to result from an action of this mutation in the retinula cells. We have estimated when the functioning of this gene or its product is required for normal visual system development by using genetic mosaicism induced by somatic recombination and temperature shifts of a temperature-sensitive mutation at this locus. Both methods point to a period in the mid-third instar, suggesting that early events in the formation of ommatidia and/or late events in the program of retinal cells are affected. Application of a new histological stain for developing axons indicates that individuals heterozygous for Glued exhibit abnormalities in the retinula fiber projection by the late third instar. Thus, the adult phenotype is not solely the result of later cellular degeneration or rearrangement. Beneath M+ Gl+ clones which encompass the entire eye were found optic lobe abnormalities with features not seen in either other mosaics or Gl heterozygotes. The possibility that these abnormalities result from temporal asynchrony in the development of eye and and optic lobe in these individuals is discussed and the results of attempts to test this hypothesis are presented.     

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.     

Reversed-phase high-performance liquid chromatographic analysis of thiamine phosphate esters at subpicomole levels

Separation and determination of thiamine phosphate esters were achieved by reversed-phase high-performance liquid chromatography (hplc) after conversion to corresponding thiochrome esters. The elution order was thiochrome triphosphate, thiochrome pyrophosphate, and thiochrome monophosphate by a system composed of 25 mm potassium phosphate buffer (pH 8.4) and 2.5% N,N-dimethylformamide. The minimum amount reproducibly detected was 0.05 pmol for each thiochrome phosphate. Thiamine phosphate esters in rat tissues were successfully determined by the reversed-phase hplc after alkaline oxidation of the tissue extract, which resulted in a good agreement in their contents to those obtained by the straight-phase hplc previously reported.     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

The influence of substitution at C24 on the calcium-binding protein-stimulating activity of vitamin D metabolites in chick embryonic duodenum

The production of calcium-binding protein, in vitro, by embryonic chick duodenum has been used to assess the potency of vitamin D compounds. The introduction of an hydroxyl on 1-, 25-, or 24R-position enhanced biological activity while the introduction of both 1α- and 25-hydroxyls produced maximal activity. However 24R-hydroxylation of 1,25-dihydroxyvitamin D₃ diminished activity. The vitamin D₂ side chain on 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D did not greatly diminish activity in contrast to the fact that the vitamin D₂ compounds are 10% as active as the vitamin D₃ compounds in vivo in the chick. These results support the idea that the target organs of the chick do not discriminate against the vitamin D₂ side chain and that the discrimination in this species is at the level of metabolism.     

Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K ( WRK ). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK . SBP was a strong protector, displaying a dissociation constant (Kd) of 3 microM with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 +/- 700 M-1 cm-1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.