Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N''-tetraacetic acid and dithiothreitol.

Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.     

Individual sarcomere length determination from isolated cardiac cells using high-resolution optical microscopy and digital image processing.

Discrete sarcomere lengths have been determined from dynamically contracting isolated cardiac cells with a high-speed, high-resolution direct optical imaging system. Calcium-tolerant cardiac cells from the rat are isolated by perfusion with collagenase and hyaluronidase. Individual sarcomere lengths can be determined by directly imaging the cell's striation pattern onto a solid-state charge-coupled device (CCD) detector interfaced with a digital computer. The precision of detection in a real light microscopic optical system is discussed in relation to the type of image detector, optical contract enhancement techniques, and digital image processing. The optical performance of the direct striation pattern image apparatus has been determined empirically with test grids under standard bright-field and Nomarski-differential interference contrast (DIC) conditions for application to real muscle imaging. Discrete striation positions of isolated cells have been detected and followed with high precision during phasic contraction-relaxation cycles down to average sarcomere lengths as short as 1.43 +/- 0.053 microns. The maximum rates of contraction and relaxation are rapid and synchronous in time course along the length of the cell. These results indicate that direct optical imaging can provide an accurate means to monitor discrete striations and sarcomere lengths along the length of Ca2+-tolerant heart cells.     

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors

The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 microM, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., & Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., & Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of gamma-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence

A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.     

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.     

Synthesis of rat hepatic zinc thionein in response to the stress of sham operation

Following sham operation for adrenalectomy, a dramatic 30-fold increase in rat hepatic zinc thionein occurs, peaking at 18 hours after surgery. Hepatic cytosolic and serum zinc levels rise concomitantly with zinc thionein. Copper in hepatic thionein and cytosol rises only slightly and serum copper not at all during the period of observation. In the period 18 to 48 hours after surgery the content of hepatic zinc thionein decreases with a $\text{t}\text{1}\text{2}$ of 16.4 hours.Pretreatment with cycloheximide (1 mg/kg b.w.) two hours before surgery inhibits the rise in zinc thionein by 52%, the rise in cytosolic zinc by 56%, and actually causes a decrease in serum zinc by 33%. Pretreatment with the α-adrenergic receptor blocker, phetolamine (10 mg/kg b.w.), or the β-adrenergic receptor blocker, propranolol (10 mg/kg b.w.), 30 minutes before surgery also inhibited the rise in zinc thionein (82% and 60%, respectively) and cytosolic zinc (75% and 47%, respectively), and decreased serum zinc (38% and 44%, respectively) 19 hours after surgery.Treatment with corticosterone (40 mg/kg b.w.) alone or epinephrine (1–20 μg/kg b.w.) alone did not alter hepatic zinc thionein levels 18 hours late, although they each caused hypozincemia and epinephrine raised cytosolic zinc levels. Treatment with corticosterone and epinephrine together did, however, raise zinc thionein levels 3.2-fold (P<0.02).These experiments are consistent with the hypothesis that adrenal hormones are involved in the regulation of zinc metabolism, and, hence, zinc thionein in levels in rat liver following the stress of sham operation.     

Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.     

Induction of tyrosine aminotransferase by pyridoxine in Drosophila hydei

Salivary glands incubated in various concentrations of pyridoxine (Vitamin B₆) show increasing tyrosine aminotransferase (TAT) activity at concentrations up to 10^–5 M and then decreasing activity up to 10^–2 M but in all cases the activity is greater than that of the controls. This increase in activity is demonstrable for up to 6 h, the longest period tested, and is dependent on the synthesis of new mRNA. A similar increase in TAT activity is observed in salivary glands subjected to heat shock. Antibodies prepared against purified tyrosine aminotransferase precipitate a peptide of the same molecular weight (40 KD) as that induced by pyridoxine.