Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine α s1-casein

Summary The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with _s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the aminp acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of _s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the _s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No cleat consensus sequence of amino acid residues surrounding the cleavage sites could be identified.Offprint requests to: G. G. Pritchard     

Graviresponsiveness of surgically altered primary roots of Zea mays

We examined the gravitropic responses of surgically altered primary roots of Zea mays to determine the route by which gravitropic inhibitors move from the root tip to the elongating zone. Horizontally oriented roots, from which a 1-mm-wide girdle of epidermis plus 2-10 layers of cortex were removed from the apex of the elongating zone, curve downward. However, curvature occurred only apical to the girdle. Filling the girdle with mucilage-like material transmits curvature beyond the girdle. Vertically oriented roots with a half-girdle' (i.e. the epidermis and 2-10 layers of the cortex removed from half of the circumference of the apex of the elongating zone) curve away from the girdle. Inserting the half-girdle at the base of the elongating zone induces curvature towards the girdle. Filling the half-circumference girdles with mucilage-like material reduced curvature significantly. Stripping the epidermis and outer 2-5 layers of cortex from the terminal 1.5 cm of one side of a primary root induces curvature towards the cut, irrespective of the root's orientation to gravity. This effect is not due to desiccation since treated roots submerged in water also curved towards their cut surface. Coating a root's cut surface with a mucilage-like substance minimizes curvature. These results suggest that the outer cell-layers of the root, especially the epidermis, play an important role in root gravicurvature, and the gravitropic signals emanating from the root tip can move apoplastically through mucilage.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

Fluid dynamics and microscale chemical movement in the chemosensory appendages of the lobster, Homarus americanus

Every chemosensory structure has a boundary layer surroundingit through which chemical signals must pass before contactingreceptor cells. Fluid motion in this boundary layer is slowand odor movement is mainly by diffusion. The boundary layerstructure depends upon external fluid velocities and the morphologyof the appendage. High-speed (10–200 Hz) electrochemicalrecordings from microchemical electrodes were used to quantifychemical transport in the microscale environment of three morphologicallydifferent chemosensory appendages of the lobster, Homarus americanus:lateral antennule, medial antennule and walking legs. Controlledpulses of the odor tracer (dopamine) were delivered to the threeappendages at three different flow speeds (0, 3, 6 cm/s). Theamplitudes of the pulses increased with increasing flow speed,indicating that boundary layer thickness decreased with increasingflow speed. Larger pulse amplitudes were measured in the walkinglegs than in the lateral or medial antennules at all flow speeds.In addition, larger amplitudes were recorded in the medial antennulethan the lateral antennule. Changes in pulse amplitude withincreasing flow speed were larger than changes in pulse duration.These results demonstrate that pulse amplitude is affected morethan pulse duration by boundary layer thickness and that themorphology of the receptor strucure helps determine the structureof signals arriving at receptor cells. This may explain whyanimals have adopted sampling strategies that reduce boundarylayer thickness.     

Role of sulfhydryl groups in benzoquinone-induced Ca2+ release by rat liver mitochondria

Incubation of rat liver mitochondria with benzoquinone derivatives in the presence of succinate plus rotenone has been shown to cause NAD(P)H oxidation followed by Ca2+ release. Further investigation revealed: (1)p-Benzoquinone-induced Ca2+ release was not initiated by a collapse of the mitochondrial membrane potential. However, Ca2+ release and subsequent Ca2+ cycling caused limited increased membrane permeability. (2) p-Benzoquinone-induced NAD(P)H oxidation and Ca2+ release were prevented by isocitrate, 3-hydroxybutyrate, and glutamate but not by pyruvate or 2-oxoglutarate. (3) Inhibition of pyruvate and 2-oxoglutarate dehydrogenases by p-benzoquinone was attributed to arylation of the SH groups of the cofactors, CoA and lipoic acid. Isocitrate dehydrogenase was also inhibited by p-benzoquinone, but the cofactors NAD(P)H and Mn2+ protected the enzyme. Glutamate dehydrogenase was not inhibited by p-benzoquinone. (4) Arylation of mitochondrial protein thiols by p-benzoquinone was associated with an inhibition of state 3 respiration, which was attributed to the inactivation of the phosphate translocase. In contrast, state 4 respiration, and the F1.F0-ATPase and ATP/ADP translocase activities were not inhibited. It was concluded that inhibition of mitochondrial NAD(P)H dehydrogenases by arylation of critical thiol groups will decrease the NAD(P)+-reducing capacity, and possibly lower the NAD(P)H/NAD(P)+ redox status in favor of Ca2+ release.     

Toxicity and partial structure of a hepatotoxic peptide produced by the cyanobacterium Nodularia spumigena Mertens emend. L575 from New Zealand.

A clonal isolate, termed L575, of the filamentous brackish-water cyanobacterium Nodularia spumigena Mertens emend. was found to produce a potent hepatotoxic peptide (50% lethal intraperitoneal dose for the mouse, 60 micrograms/kg) with chemical and toxicological properties similar to those of the hepatotoxic heptapeptides produced by other freshwater planktonic cyanobacteria. The isolate was made from a water sample collected in Lake Ellesmere, New Zealand, in 1980. The toxin, isolated and purified by high-performance liquid chromatography (HPLC) and analyzed by HPLC amino acid analysis, contained glutamic acid, beta-methyla-spartic acid, and arginine units in equivalent amounts. The fast-atom-bombardment mass spectrum of the toxin indicated the molecular weight to be 824. Batch cultures of strain L575 showed that the toxin content varied between 1.96 and 2.99 mg/g of cells and that a positive correlation between toxin content and chlorophyll a, but not biomass, was present.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

M?ssbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe M?ssbauer spectroscopy. The M?ssbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.