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.     

The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

Changes in lung volume and deflation stability in hyaline membrane disease

Total lung capacity (TLC), inspiratory capacity, functional residual capacity, and deflation stability of prematurely delivered Macaca nemestrina primates were measured serially during development of, and recovery from, hyaline membrane disease (HMD) to relate changes in lung volumes to changes in deflation stability. Gestational age-matched primates that did not develop HMD served as controls. TLC, measured by N2 washout, fell at 2-12 h of age (P less than 0.0001) in animals with HMD and remained lower than controls for at least 48 h (P less than 0.005). However, deflation stability, defined as the fraction of TLC remaining upon deflation to 10 cm H2O, improved from 2 to 12 h of age (P less than 0.001). Postmortem studies confirm the measurements of TLC and deflation stability and provide evidence that interstitial thickening and obstruction of air spaces with debris may be partially responsible for the observed changes in TLC in primates that develop HMD. It has been assumed that TLC is reduced in HMD because of atelectasis from elevated alveolar surface tension, but the sequential measurements in these animals suggest that other mechanisms also contribute.     

Attenuation of noradrenergic neurotransmission by the thromboxane synthetase inhibitor,UK 38,485

The purpose of this study was to determine the effects of chronic administration of the thromboxane synthetase inhibitor, UK 38,485, on noradrenergic neurotransmission. Male Sprague Dawley rats (n=14) were treated once daily with either UK 38,485 (100 mg/kg; n=7) or the vehicle of UK 38,485 (olive oil; n=7) by gavage. The dose of UK 38,485 chosen was sufficient to inhibit $\text{ex vivo}$ platelet TXB₂ production by >90% for 24 hours. One week into the treatment animals were prepared for $\text{in situ}$ perfusion of their mesenteric vascular beds. Vasoconstrictor responses to both exogenous norepinephrine and periarterial nerve stimulation were determined both before and during an infusion of angiotensin II (9ng/min) into the superior mesenteric artery. UK 38,485 significantly (P<0.02) attenuated the vascular response to periarterial nerve stimulation without altering the vascular response to either norepinephrine or angiotensin II. UK 38,485 did not influence the baseline perfusion pressure, the mean arterial blood pressure or the potentiation of neurotransmission by angiotensin II. These data indicate that in the $\text{in situ}$ rat mesentery UK 38,485 attenuates the release of neurotransmitter from sympathetic nerve terminals.