Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.     

A review of some physiological and evolutionary aspects of body size and bud size of Hydra

Green hydra with endosymbionts are smaller than brown asymbiotic ones. Regeneration experiments, mitotic index studies on algal and hydra tissue, and evidence for consumption and expulsion of algae are reviewed and it is suggested that larger green hydra have more difficulty controlling algal increase than smaller ones and that hydra have an upper size limit for maintenance of stable symbioses. A mathematical model is discussed which starts with simple physiological assumptions about hydra and generates field testable conclusions about how body and bud size, and reproductive rates depend on food particle size, quantity and temporal distribution. Unlike most analytic ecological-evolutionary models, this one integrates physiology, ecology and evolution without needing simplifying assumptions.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus.

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.     

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the -helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to -polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D) and perpendicular (D) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P to L/P 15 results in a decrease of the rotational diffusion coefficient D from 3.4 × 10⁷ to 1.9 × 10⁷ s^–1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters P₂ and P₄, and the rotational diffusion coefficient D of the fluorescent probe. The order parameters P₂ and P₄ increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35. The rotational diffusion coefficient D of the DPH-propionic acid probe decreases on incorporating M13 coat protein, in accordance with the ESR results. It is concluded that M13 coat protein in the -monomeric state is not able to produce a long living lipid boundary shell and consequently an immobilization of the lipids. An overall effect on the lipids is induced, resulting in a reduction in the dynamics and an increase in average lipid order. The hydrophobic region of M13 coat protein is proposed to perfectly match the lipid bilayer, resulting in a relatively small distortion of the bilayer structure of the lipid system.     

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

Identification, characterization, and homologous up-regulation of latent (cryptic) receptors for tumor necrosis factor-alpha in rat liver plasma membranes

A population of latent (cryptic) receptors for tumor necrosis factor-alpha (TNF) has been characterized in the rat liver plasma membrane (PM). 125I-TNF bound to high (Kd = 1.51 +/- 0.35 nM) and low (Kd = 13.58 +/- 1.45 nM) affinity receptors in PM. Solubilization of PM with 1% Triton X-100 prior to incubation with 125I-TNF increased both high affinity (from 0.33 +/- 0.04 to 1.67 +/- 0.05 pmol/mg of protein) and low affinity (from 1.92 +/- 0.16 to 7.57 +/- 0.50 pmol/mg of protein) TNF binding without affecting the affinities for TNF. Digestion of intact PM with chymotrypsin abolished most of the TNF binding capacity of PM. However, substantial binding activity was recovered by solubilization of chymotrypsin-treated PM with 1% Triton X-100, suggesting the presence of a large latent pool of TNF receptors. The affinities of the high and low affinity sites recovered from chymotrypsin-treated membranes were similar to those of intact PM. Affinity labeling of receptors whether from PM, solubilized PM, or membranes digested with chymotrypsin and then solubilized resulted in cross-linking of 125I-TNF into Mr 130,000, 90,000, and 66,000 complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, 125I-TNF binding to control and TNF-pretreated membranes was assayed. Specific binding was increased by pretreatment with TNF (p less than 0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF.     

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.     

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle

Summary Changes in the distribution of the in vitro uptake of ¹²⁵I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 m, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of ¹²⁵I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of ¹²⁵I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of ¹²⁵I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.