Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.     

Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis

The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10,200 +/- 700 for ovomucoid, 17,800 +/- 400 for ovoinhibitor and 16,400 +/- 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.     

The functional molecular weights of factor VIII activities in whole plasma as determined by electron irradiation

Activities of the various components of the human factor VIII complex in citrated and heparinized human plasma have been determined following radiation inactivation of the plasma in a high energy electron beam at -135 degrees C in order to determine the molecular size of the functional units. In citrated and in heparinized plasma the functional size of VIII:C was 120,000 +/- 9,700 and 140,000 +/- 10,000, respectively. Taken together with previously published data, these results suggest that VIII:C exists in plasma as a dimer of noncovalently bonded functional subunits. The size of the functional unit of the ristocetin cofactor of the factor VIII complex was determined as being approximately 330,000 in both citrated and heparinized samples. Immunological assays for VIII:C (inhibitor neutralization assay), the VIII:C antigen, and the VIII:vWF-related antigen suggest that these may not be reliable under conditions favoring the activation and inactivation of factor VIII components by thrombin or other proteases.     

Endogenous steroid concentrations in human breast tumours determined by high-resolution mass fragmentography (Short Communication)

A pilot study of the endogenous steroid concentrations in human breast tumours was performed. The technique of high-resolution molecular-ion monitoring during combined g.l.c.-mass spectrometry was used to determine oestrone, oestradiol-17beta and oestriol in concentrations above 1ng/g wet wt. of tissue, and dehydroepiandrosterone, testosterone, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) and 3beta-hydroxy-5alpha-androstan-17-one in concentrations exceeding 5ng/g, in extracts of five primary breast tumours.     

Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.

Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.     

Modulation of erythrocyte acetylcholinesterase by cardiolipin: Effect on subunit coupling revealed by irradiation inactivation

The functional molecular weights of two kinetically distinct forms of bovine erythrocyte acetylcholinesterase were determined by irradiation inactivation. Whereas both forms have similar molecular weights by hydrodynamic measurements and contain 33 molecules of cardiolipin, the functional molecular weight of form α (140,000) was found to be twice that of form β (73,000). As form β is derived from form α by treatment with high salt concentration in alkaline Ca²⁺-chelating conditions, a procedure which is considered to disrupt the functional association of a Ca²⁺-cardiolipin complex with the enzyme, it is suggested that cardiolipin mediates the energy transfer between enzyme subunits, thereby modulating the kinetic properties of the lipoprotein.     

The solubility of fibrinogen in solutions containing dextrans of various molecular weights (Short Communication)

The effect of dextrans of various molecular weights on the solubility of fibrinogen was investigated. The results indicate that the prime effect responsible for the observed changes of solubility was steric exclusion. This applies over the whole molecular-weight range of dextrans used, i.e. from 5700 to 76000.     

The functional size of acyl-coenzyme A (CoA):cholesterol acyltransferase and acyl-CoA hydrolase as determined by radiation inactivation

Frozen rat liver microsomes and rough endoplasmic reticulum were irradiated with high energy electrons. The surviving enzymatic activity of acyl-CoA:cholesterol acyltransferase and activity for esterification of 25-hydroxycholesterol decreased as a simple exponential function of radiation exposure, leading to a target size of 170-180 kDa. The loss of acyl-CoA hydrolase activity with a radiation dose was complex and resolved as a 45-kDa enzyme associated with a large inhibitor. It is interpreted that acyl-CoA hydrolase is the acyl-CoA-binding component and the inhibitor is the cholesterol-binding component of acyl-CoA:cholesterol acyltransferase.     

pH-controlled hydrogen-bonding (Short Communication)

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)(-1) where p=2.303 (pK(a)-pH), pK(a) being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pK(a)+/-logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.     

The potentiation of growth hormone by asparagine and tryptophan (Short Communication)

Asparagine potentiates the growth-promoting effect of bovine growth hormone in rats when injected 3h after the latter, but not earlier. Tryptophan potentiates only when injected simultaneously with the hormone. The possible mode of action of growth hormone, and the likelihood that asparagine is a limiting factor in the growth of animals, are discussed.     

Molecular size of opiate (enkephalin) receptors in neuroblastoma-glioma hybrid cells as determined by radiation inactivation analysis

Opiate receptor binding decayed exponentially in mouse neuroblastoma-rat glioma (NG108-15) hybrid cell preparations following exposure to increasing doses of ionizing radiation (0.2 to 7.0 Mrads; 2.0 Mrads/min). Target size analysis revealed that [3H][D-Ala2, D-Leu5]enkephalin (agonist) and [3H]naloxone (antagonist) bound specifically to a component with an apparent molecular size of 200,000 +/- 20,000. Lyophilization of cells for the irradiation procedure did not significantly alter receptor affinity or binding capacity for these ligands. Furthermore, the loss of opiate receptor binding in irradiated cell samples could not be attributed to reduced receptor affinity since increasing concentrations of radiolabeled ligand failed to reverse the inhibition; nonspecific binding decreased only slightly under identical experimental conditions. The value of determining molecular size by radiation inactivation analysis was confirmed by showing that apparent target sizes for two representative lysosomal enzymes (beta-galactosidase and alpha-mannosidase) were consistent with results obtained previously using conventional methods. Thus, the data suggest that the ligand binding component of delta-opiate (enkephalin) receptors in NG108-15 cells has a minimum functional size of approximately 200,000.     

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.     

Proteolysis of rabbit immunoglobulin M by papain (Short Communication)

1. Digestion of rabbit immunoglobulin M (IgM) by papain for 5h, in the absence of reducing agent, gives Fabmu and Fc(5)mu fragments in high yield. Shorter periods give fragment Fc(5)mu with one or more Fabmu fragments still attached. 2. Reducing agent in the absence of a denaturant cleaves rabbit IgM into half-subunits, each containing one mu chain and one light chain. Digestion by papain in the presence of such a reducing agent destroys the Fcmu domains.     

Inhibition of mitochondrial phosphate transport by p-diazobenzenesulphonate (Short Communication)

The impermeant protein-modifying reagent p-diazobenzenesulphonate inhibits phosphate transport in rat liver mitochondria. The rate of inhibition is increased by the presence of oxidizable substrate, and decreased by uncoupling agents. These results are compared with the results on inhibition of phosphate transport by N-(N-acetyl-4-sulphamoylphenyl)maleimide (Klingenberg et al., 1974).     

Acceleration of gluconeogenesis from lactate by lysine (Short Communication)

l-Lysine (2mm) causes an increase (mean 60%) in the rate of gluconeogenesis from lactate in isolated liver cells. The effect is of a catalytic nature. No other amino acid has the same effect, though ornithine is slightly active. The effect is additional to the stimulatory effects of oleate and of dibutyryl cyclic AMP.     

Functional lysosomal hydrolase size as determined by radiation inactivation analysis.

Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.     

The effect of l-ascorbate on catecholamine biosynthesis (Short Communication)

l-Ascorbate stimulates the enzymic hydroxylation of phenylalanine in vitro by recycling tetrahydrobiopterin, which reduces O₂ utilized in the reaction. It is suggested that ascorbate might have a similar function in vivo; this would explain the apparent regulation of tyrosine hydroxylase and tryptophan hydroxylase activities by this vitamin.     

The reaction of N-acetylneuraminate lyase with chloropyruvate (Short Communication)

Chloropyruvate, like bromopyruvate, rapidly inactivates N-acetylneuraminate lyase at pH7.2. At 5 degrees C, 0.5mm-chloropyruvate reacted with the enzyme about ten times as fast as bromopyruvate. In contrast, at pH6.0 and 9 degrees C, chloropyruvate reacted with N-acetylcysteine seven times more slowly than bromopyruvate. A brief (2min) incubation of the enzyme with 1.0mm-chloro[(14)C]pyruvate gave an inactive enzyme in which 4.5 cysteine residues were alkylated per molecule of enzyme. This corresponded to the number of [(14)C]pyruvate residues (3.7) bound to the enzyme by borohydride reduction of the [(14)C]-pyruvate complex, and confirmed the previous suggestion that there is one ;essential' cysteine residue per active site. It is suggested that, for this enzyme, chloropyruvate can be selectively used to alkylate the active-site residues, whereas bromopyruvate cannot. The apparent molecular weight of the enzyme prepared by two slightly different methods was approx. 100000 or 250000. The latter value has been used to calculate the number of pyruvate residues bound to the enzyme.     

Specificity of anti-nucleoside antibodies (Short Communication)

The method of conjugation of a nucleoside or related compound to a carrier protein may have a significant effect on the specificity of the antibodies elicited. It is demonstrated, by means of the membrane-filtration assay, that anti-isopentenyladenosine antibodies produced by the ;periodate procedure' are much more reactive with the periodate-oxidized form of the nucleoside than with the parent compound. In addition, the simplicity and specificity of the assay used suggests its use as a sensitive radioimmunoassay for this multifunctional nucleoside.