Design, synthesis, and testing of insulin hexamer-stabilizing agents.

The addition of zinc to insulin solution leads to a long-acting insulin preparation because the zinc stabilizes the less soluble hexameric form of the hormone. It is clear from the crystal structure of dizinc insulin that there is a space at the center of the hexamer, between the two zinc atoms, that could accommodate a small organic molecule. It should thus be possible to design a structure that could further stabilize the insulin hexamer by binding at this site. Computer graphic techniques have been used to design several molecules capable of forming multiple bonds to the six histidine residues surrounding the site. Synthesis and testing of one of these compounds, benzene-1,4-disulfonic acid, show a significant increase in weight-average molecular weight of insulin in solution, and control experiments with related structures suggest that this effect is due to the proposed binding mechanism.

Fat emulsion catabolism in vitro and in vivo--sex related differences.

The removal rates of an intravenously administered 10% fat emulsion (Intralipid) from plasma in male and female conscious rats are described. The plasma concentration of fat emulsion particles at various time intervals following a bolus administration (0.2 g/kg) was measured by nephelometry. At the dose employed, the removal of fat emulsion from the plasma followed first order kinetics, ie, a constant fraction was removed from the plasma per unit of time, K2 (%/min). Females exhibited a significantly greater fractional removal rate (K2) than comparably aged males (21.0 +/- 1.0 vs 15.0 +/- 1.4, p less than 0.05). Postheparin lipoprotein lipase, measured using fat emulsion as substrate, also was significantly greater in female rats compared with males. Our results demonstrate that, in rats, fat emulsion (Intralipid) is catabolized more rapidly in females than in males and a greater lipoprotein lipase activity in female rats may be the causative factor.

Aggregation of wool keratin intermediate filament proteins.

The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.

Subunit structure of oyster paramyosin.

Paramyosin from the oyster Crassostrea commercialis was studied by equilibrium sedimentation. In non-denaturing solvents the minimum molecular weight is 208000. Dissociation into subunits requires complete disruption of the alpha-helix. This occurs at pH7 in guanidine hydrochloride solutions of concentration greater than 7m in the presence of a disulphide-bond-reducing agent. Solutions of the protein in concentrated guanidine hydrochloride are polydisperse and contain species of low molecular weight (approx. 25000) comprising approx. 5% to 10% of the protein. The molecular weight of the main component is estimated to be 97000 and the paramyosin molecule contains two of these subunits. From the present observations no decision can be made as to whether or not the small component (or components) represents part of the paramyosin molecule. Preferential binding of guanidine hydrochloride to the extent of 0.13g./g. of protein was shown in solutions of paramyosin in 7.85m-guanidine hydrochloride.

Stability of segments of rabbit alpha-tropomyosin.

Rabbit alpha-tropomyosin was cleaved into two pieces at the cysteine residue of each chain. The products were separated by chromatography and characterized by amino acid analysis, molecular weight determination in benign and denaturing solvents, optical rotation and circular dichroism. When the cleavage reaction was carried out under mild conditions which preserve the two-chain structure there was considerable loss of alpha-helix in each segment. Thermal stability studies, monitored by optical rotation and circular dichroism, showed that the transition temperature of the N-terminal fragment at pH 7.6 was approximately 17 degrees C higher than that of the C-terminal fragment. In acid solutions there is little difference in the thermal stability of the two segments. The least stable part of the molecule is concluded to be between residues 133 and 205 and this includes the troponin-binding site. The relative stabilities found for segments of rabbit alpha-tropomyosin differ from recent published conclusions and this may be a result of the different methods used to study the loss of the alpha-helical conformaton. The two tropomyosin fragments, unlike the parent tropomyosin, do not inhibit actomyosin adenosinetriphosphatase when mixed with troponin. The fragments did not show any of the aggregation properties of tropomyosin and did not combine with actin. The N-terminal fragment did not complex with troponin but there was some evidence for an interaction between the C-terminal fragment and troponin.

The number of polypeptide chains in the rod domain of bovine epidermal keratin.

The physical properties of the alpha-helix enriched particle obtained by limited proteolysis of epidermal keratin have been examined. Molecular weights in benign and dissociating solvents, hydrodynamic measurements and chemical cross-linking experiments indicate that the particle has four polypeptide chains. It is proposed that the helical particle is assembled from a pair of two-stranded alpha-helical ropes.

The conformational stabilities of tropomyosins.

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the alpha-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin. Differences in stability were found between tropomyosins from different muscles of the same species as well as between species, no two tropomyosins giving the same denaturation profiles. The invertebrate tropomyosins showed a wider range of stabilities, that from scallop striated muscle being far more easily denatured than all the others. No correlation was found between the stability of tropomyosin and the type of regulatory system of the muscle. A comparison of the results from vertebrate and invertebrate species suggests that there has been no selection for proteins of higher or lower stability during the evolutionary time scale.

Sedimentation equilibrium studies on protein from kookaburra beak.

Fractionated samples of the soluble S-carboxymethyl proteins from kookaburra beak (Frenkel and Gillespie 1976) were examined by equilibrium sedimentation. The molecular weight was found to be 11,300 when the photoelectric scanning absorption optical system was employed and 13,700 when Rayleigh interference optics were used. Possible explanations for this difference are considered and it is concluded that it must arise from heterogeneity of the protein. Optical rotatory dispersion measurements indicate that the proteins probably exist as random coils in dilute aqueous buffer.

Structural studies on the microfibrillar proteins of wool: characterization of the alpha-helix-rich particle produced by chymotryptic digestion.

The alpha-helix-rich particle produced by chymotryptic digestion of the reduced and alkylated microfibrillar proteins of wool was characterized by physicochemical methods. The preparations were homogeneous with respect to size and the particle molecular weight was found to be 50 200 +/- 2 000. Hydrodynamic methods indicated a length of about 20 nm for the particle. The properties of the particle, derived from two methods of isolation of the microfibrillar proteins, were identical and were also independent of the type of wool used. From a consideration of the molecular weight in denaturing solvents and from cross-linking experiments with dimethyl suberimidate a four-chain structure, consisting of a pair of double-stranded alpha-helices, is proposed for the particle.

Structural studies on the microfibrillar proteins of wool. Interaction between alpha-helical segments and reassembly of a four-chain structure.

The alpha-helix-rich particle of Mr 50 200, derived by limited alpha-chymotryptic digestion of the solubilized microfibrillar proteins from wool alpha-keratin, consists mainly of polypeptide-chain segments of Mr 12 500 (fraction ChC) and 25 000 (fraction ChB). The 12 500-Mr segments are of two types (I and II), which are derived from different polypeptide chains of the microfibrillar complex. Each of these type-I and type-II segments partially self-associates in benign solvents to form either dimers or tetramers. When mixed, the two segments show changes in physical properties (alpha-helix content, difference spectra and molecular weight) indicative of complex-formation. The maximum changes occur when the two segments are mixed in an equimolar ratio. Complexes isolated after rapid dialysis of mixtures from 8 M-urea solution were examined by various methods. A tetrameric structure is the main product formed in all cases, and the maximum amount of tetramer is obtained from equimolar mixtures of the type-I and type-II polypeptides. When urea is removed by dialysis from the unfractionated 12 500-Mr segments (fraction ChC) or from the alpha-helix-rich particle itself, a similar complex of Mr 50 000 is formed. The physical properties of these reconstituted entities (alpha-helix content, molecular weight, thermal stability and exposure of tyrosine residues) are similar to those of the original alpha-helix-rich particle. Cross-linking experiments with dimethyl suberimidate are in agreement with a four-chain complex for the reassembled structures. A pair of double-stranded alpha-helices is proposed for the particle, and is considered to be an integral part of the microfibrillar complex in wool alpha-keratin.

Biotransformation of sevoflurane in dogs and rats.

Sevoflurane, 3% and 4% in oxygen was administered to four dogs for 3 hours. Sevoflurane was metabolized to inorganic fluoride and hexafluoroisopropanol. Serum fluoride concentrations reached peak values during 2 to 3 hours into anesthesia and averaged 18.5 micrometer/L (n = 2) and 20.0 +/- 4.8 (mean +/- SD) micrometer/L (n = 4) following 3% and 4% sevoflurane exposure, respectively. They returned to normal values within 24 hours after anesthesia. Hexafluoroisopropanol was excreted in the urine as glucuronide conjugate. Its elimination was essentially complete within 48 hours after the end of exposure to sevoflurane. During inhalation of 4% sevoflurane, blood concentration of the anesthetic reached an average apparent steady state of 0.765 +/- 0.10 micrometer/L (n = 4). No anesthetic was detected in blood 24 hours after this exposure. Rats were anesthetized with 2% sevoflurane for 2 and 4 hours. Immediately after anesthesia, observed mean (n = 6) serum fluoride concentrations were 2.9 +/- 0.5 micrometer/L and 2.5 +/- 0.6 micrometer/L, respectively. Hepatic microsomal enzyme induction produced by pretreatment with either phenobarbital or polychlorinated biphenyls (PCBs) resulted in an approximately 5-fold increase in serum fluoride concentrations following anesthesia with sevoflurane when compared to noninduced rats exposed to sevoflurane. A comparison of serum fluoride concentrations between the rat and dog indicates that the amount of sevoflurane metabolized is lower in the rat than in the dog, and the fluoride concentrations observed in both animal species during sevoflurane anesthesia are not expected to produce nephrotoxicity.

Hemodynamic and metabolic responses to repeated blood sampling in the rat.

Repeated blood sampling (0.2 ml) in the conscious rat during the course of 10 cardiac index measurements using the Fick procedure did not alter the cardiac index as measured initially (285 ml x min-1 x kg-1). However, oxygen consumption and hematocrit were reduced 7-19% and 4-14%, respectively. Replacement of blood removed during sampling with donor blood prevented these responses, but also led to reduced cardiac index and arterial oxygen content, 22-28% and 10-21%, respectively. In additional studies in anesthetized rats, hemorrhage (25 ml/kg) increased plasma K+ by 29% and reduced plasma Na+ by 3%, suggesting that compensatory fluid replacement originated in cells as well as interstitium. This fluid replacement after blood loss helps sustain normal systemic hemodynamics, but blood loss can produce metabolic alterations that should be taken into account in any biochemical study. Although metabolic alterations can be prevented by replacing lost blood with donor blood, cardiopulmonary function may be adversely affected.

Improved carbohydrate tolerance in fibre-fed rats: studies of the chronic effect.

The effect of chronic consumption of diets containing either different sources of dietary fibre (8% guar, 8% pectin, or 8% multifibre) or low carbohydrate upon carbohydrate tolerance was examined in rats. Weight gain was significantly lower throughout the entire 28-day study period with the guar group and after 20 days with the multifibre group. When tested with a liquid meal (1 g sucrose/kg body weight) after 14 days on the diets, only the guar rats had significantly lower fasting and postprandial plasma glucose concentrations. After 28 days, the improved carbohydrate tolerance persisted in the guar rats and started to appear in the multifibre rats. Pectin and low carbohydrate diets had no effect upon either weight gain or carbohydrate tolerance. Consuming the fibre diets did not affect jejunal sucrase activities. Jejunal glucose uptake activity was significantly diminished when measured in fasting guar rats while postprandially activities were similar to controls. Jejunal Na-K-ATPase activities in fasting and postprandial guar rats were not related to changes in glucose uptake. These studies confirm that only certain types of dietary fibre improve carbohydrate tolerance and suggest that reduced weight gain and altered intestinal glucose uptake are factors involved in the chronic fibre effect.

Disposition kinetics of ethylene oxide, ethylene glycol, and 2-chlorethanol in the dog.

The disposition kinetics of ethylene oxide, ethylene glycol, and 2-chloroethanol were studied following their intravenous administration to beagle dogs. Plasma concentration of ethylene oxide was found to decline exponentially with a mean rate constant of 0.024 +/- 0.008 min-1 (mean +/- SD) and total body clearance of 20.0 +/- 5.2 ml/kg X min. Ethylene oxide was found to be metabolized mainly to ethylene glycol, which had a mean plasma half-life of 221.0 +/- 77.7 min and a total body clearance of 2.13 +/- 0.58 ml/kg X min. Between 7 and 24% of intravenously administered ethylene oxide was eliminated in the urine as ethylene glycol within 24 h. The elimination half-life and clearance values for 2-chlorethanol were 40.8 +/- 5.7 min and 10.3 +/- 1.7 ml/kg X min, respectively. The pharmacokinetic data gathered in the present investigation suggest that ethylene glycol rather than 2-chloroethanol is the major metabolite of ethylene oxide in the dog.

Determination of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma using solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization.

Phenethyl isothiocyanate is unstable in aqueous media and at low pH, and rapidly degrades to phenethylamine. Concentrations of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma, were determined utilizing solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization using acetone as the reagent gas. Deuterated d5-amphetamine was used as an internal standard. After extraction, phenethylamine and d5-amphetamine were derivatized using MBHFBA. Ions monitored for d5-amphetamine were m/z 337 and 338; and for phenethylamine were m/z 318 and 319. Precision and accuracy were studied using control solutions prepared in naive dog plasma (80 and 300 ng/ml). Intra-day variability was determined using six replicates of each control solution analyzed on a single day. The relative standard deviation for the 80 ng/ml control was 12.9% and for the 300 ng/ml it was 12.1%. Relative accuracy was 10.9% for the low control and -4.1% for the high control. Inter-day variability was determined over a 6-day period. For the 80 and 300 ng/ml control solutions, the relative standard deviations were 15.8 and 9.1%, respectively, and relative accuracy values were 10.1 and -5.2%, respectively. Standard curves were prepared in naive dog plasma and were linear over the range of phenethylamine assayed (10-500 ng/ml). The results of this study indicate that the proposed method is simple, precise, accurate and sensitive enough for analysis of large numbers of plasma samples.