"Metallothionein-like" metal complexes in angiosperms; their structure and function

Evidence for the presence of the metal-binding protein metallothionein, MT, in higher plants is equivocal. Although a number of MT-like metal complexes have been isolated from plants, the chemical structures of most of these compounds have not been fully elucidated. Recently a novel class of plant peptides, poly (γ-glutamylcysteinyl) glycines, (γEC)_nG, have been discovered. These peptides bind metal ions, and in the presence of such ions the amount of (γEC), G in plant cells increases. The presence of peptide bonds through the γ-carboxyl group of glutamate, rather than the α-carboxyl group, suggests that these peptides are not encoded by structural genes but are the products of biosynthetic pathways. Cells which are resistant to supra-optimal concentrations of certain metal ions over-produce (γEC)_n G. (γEC)_n G. may be functional analogues of MT. Whether or not some plants also produce MT is an important question which remains to be answered.     

Use of P NMR to Assess Effects of DNP on ATP Levels in Vivo in Barley Roots

Previous work has shown that undissociated 2,4-dinitrophenol (DNP) both increases the permeability of roots to ions and alters the membrane lipids of barley roots. Anionic DNP is the main entrant form but has no effect on permeability or on the membrane lipids. The amount of anionic DNP taken up by the roots is sufficient, that were it in free solution in the cytoplasm, the DNP would uncouple oxidative phosphorylation, and thereby inhibit ATP synthesis. The present work was undertaken to assess whether DNP alters ATP levels when it is taken up by barley roots. ³¹P nuclear magnetic resonance spectra were used to monitor, in vivo, levels of ATP, cytoplasmic phosphate, vacuolar phosphate, and other phosphate compounds in barley roots in the presence of 10 micromolar DNP at pH 5 and pH 7. The spectra indicate that no change in the level of ATP or the cytoplasmic pH occurred in the roots in the presence of DNP for as long as 20 hours. Thus, the effects of undissociated DNP are effects directly on the root membranes and do not involve inhibition of ATP synthesis. Furthermore, the results explain why anionic DNP has no effect on ion uptake and accumulation.     

Histone segregation on replicating chromatin

We have reinvestigated the mode of segregation of preexisting histones onto replicating chromosomes. Since our previous data have indicated that only histones H3 and H4 do not appear to move from their association with the DNA strand with which they are bound until the next round of replication, we have concentrated our attention on these two histones. The strategy we have employed involved density labeling of DNA and radiolabeling of the histones of interest. Subsequently, we followed the association of histones and DNA during further rounds of DNA replication. One can make predictions concerning the nature of the association between specific histones and particular DNA strands depending on the mode of deposition. The results have confirmed our previous findings that histones segregate randomly. The possibility that such a result is a consequence of turnover of radiolabel in non-histone proteins and subsequent reutilization for histone synthesis has been tested directly. This process appears to be occurring to only a very limited extent. The implications of these conclusions for chromatin structure and gene control are discussed.     

Electron spin resonance studies of intact mammalian skeletal muscle

Samples of skeletal muscle from mice, rats and man have been examined by conventional electron spin resonance techniques. One major free-radical signal with g value 2.0036-2.004 was detected in all intact muscle samples and homogenates at 77 K whereas this signal was not seen at room temperature. Other less prominant signals were also detected. Thirty minutes of excessive contractile activity of rat hind limb muscles was found to result in a leakage of intracellular creatine kinase enzyme into the blood plasma and also produced an average 70% increase in the amplitude of the major electron spin resonance signal. These data support the hypothesis that increased free-radical activity may play some role in muscle damage caused by extensive muscular activity.     

A rabbit reticulocyte factor which stimulates protein synthesis in several mammalian cell-free systems

Rabbit reticulocyte lysate post-ribosomal supernatant is shown to stimulate protein synthesis in a variety of mammalian cell-free systems, particularly the less efficient systems, such as those from mouse liver, HeLa cells and heat-shocked L cells. This stimulation reflects an increase in the rate of initiation, and is not due to the presence of globin mRNA. The stimulatory activity is unstable to purification, but some conditions favouring stability have been identified and partial purification has been achieved. It is free of eIF-2, but possesses eIF-2B activity. Its purification properties suggest that it is distinct from previously characterized initiation factors, including eIF-2/eIF-2B complex, and its possible relationship to known initiation factors is discussed.     

Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure

The transmembrane protein bovine rhodopsin was reconstituted with egg phosphatidylcholine (PC) by using a modified detergent dilution technique employing the nonionic detergent octyl-beta-D-glucoside (octyl glucoside). Using this technique, reconstituted membranes having molar phospholipid/protein ratios between 60:1 and 255:1 were prepared. This is in contrast to the results obtained when an octyl glucoside dialysis technique was employed (Jackson, M.L. and Litman, B.J. (1982) Biochemistry 21, 5601-5608). In the latter case, the highest molar phospholipid/protein ratio that could be obtained when reconstituting rhodopsin with egg PC was approximately 50:1. Reconstituted vesicles prepared by the octyl glucoside dilution technique were examined by negative stain and freeze-fracture electron microscopy, and it was found that the vesicles were unilamellar providing the molar PC/protein ratio was below about 200:1, whereas in preparations having ratios higher than this, a significant number of the vesicles were multilamellar. The mean vesicle diameter showed no trend based on the molar PC/protein ratio within the range of 82:1 to 186:1. The mean diameters of the preparations were between 520 and 850 A. Approximately equal numbers of protein particles were observed on the concave and convex fracture faces of the freeze-fracture micrographs of the reconstituted membranes which is indicative of a symmetric distribution of the protein across the bilayer.     

Peptide products of the cleavage of bovine preprolactin by signal peptidase

Cleavage of preprolactin (pPL) by detergent-solubilized signal peptidase produced mature prolactin and two small peptides derived from the signal peptide region of the pPL molecule. The production of both peptides was dependent on functional signal peptidase; the peptides were not generated at detergent concentrations that abolished signal peptidase activity. The amount of both peptides was proportional to the concentration of signal peptidase in the assay. The appearance of both peptides was insensitive to protease inhibitors, as was signal peptidase activity. The size, labeling characteristics, and amino acid sequence of the larger peptide, peptide 1, corresponded to those of the intact signal peptide of pPL. The smaller peptide, peptide 2, lacked the carboxy terminus of the signal peptide, and was, therefore, a fragment of intact signal peptide. These results demonstrate the endoproteolytic nature of signal peptidase.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

Monoclonal antibodies to bovine milk lipoprotein lipase. Evidence for proteolytic degradation of the native enzyme

Lipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose. Polyacrylamide gel electrophoresis showed a single protein with an apparent molecular weight of 55,000 in the trailing edge of the elution profile; fractions in the leading edge contained additional proteins with molecular weights of 36,000 and 18,000-22,000. Nine monoclonal antibodies were prepared against the 55,000-dalton protein. By immunoblotting, we show that the Mr = 18,000-22,000 components share common antigen determinants with the 55,000-dalton protein, suggesting that they represent proteolytic degradation products. Incubation of partially purified lipoprotein lipase for 24 h at 37 degrees C results in breakdown of the 55,000-dalton protein with concomitant enrichment in lower Mr components; the proteolytic activity is prevented by incubating the milk with phenylmethane, sulfonyl fluoride prior to chromatography on heparin-Sepharose. This study shows the presence of milk proteases which co-purify and degrade lipoprotein lipase. We suggest that this degradation could account for part of the known instability of the enzyme.     

Orbital volume measurements in enophthalmos using three-dimensional CT imaging

The purpose of this study was to investigate enophthalmos by measuring the volume of various orbital structures using off-line computer techniques on images generated by a CT scanner. Eleven patients with enophthalmos had CT scans of the orbits consisting of 30 to 40 adjacent 1.5-mm slices. The data from the scans were analyzed on a Nova 830 stand-alone computer system using software programs that allowed measurement of total bony orbital volume, total soft-tissue volume, globe volume, orbital fat volume, neuromuscular tissue volume, and apex-to-globe distance in the horizontal plane. These data were analyzed comparing the volumes in the normal eye with the volumes in the enophthalmic eye in each patient. The analysis demonstrated a statistically significant increase in bony orbital volume in the enophthalmic eye, but the total soft-tissue volume, fat volume, neuromuscular tissue volume, and globe volume were the same as in the normal eye. The apex-to-globe distance, a measure of the degree of enophthalmos, was less in the enophthalmic eye than in the normal eye. These results suggest that in the majority of patients, the cause of posttraumatic enophthalmos is increased bony orbital volume rather than by soft-tissue loss or fat necrosis. (Several patients showed no volume discrepancies, and it is likely that cicatricial contracture is responsible for the enophthalmos in these cases.) This study suggests that the objective of surgery for correction of enophthalmos in patients with a volume discrepancy should be to decrease the volume of the bony orbit and to increase the anterior projection of the globe.