Effect of cell size, age and anatomical location on the lipolytic response of adipocytes

Studies were conducted on the norepinephrine (NE) stimulated lipolytic sensitivity of adipocytes from epididymal (Epi), perirenal (PR), subcutaneous (SC) and mesentric (M) depots from young (7–8 wk.) and adult (14–16 wk.) male rats. In the young rats dose response curves to NE were similar for Epi, PR and M depots whereas adipocytes from the SC depot showed a diminished effect over the mid-portion of the curve. This difference could not be ascribed to differences in cell size. In the adult rats glycerol release in the Epi depot in response to NE was identical to the younger rats which was in marked contrast to the other depots in which glycerol release was decreased in comparison to the younger animals. This decreased responsiveness was probably largely a result of age and not changes vn adipocyte size within a given depot. In these older rats, glycerol release was greatest in the Epi cells, least in the SC and M depots, and intermediate in PR. When young rats were subjected to a 72-hour fast, loss of triglyceride per cell was the same in all depots as predicted by the $\text{in vitro}$ data whereas in old rats (610 g), triglyceride loss was proportional to cell size with Epi ≥ PR > SC ≥ M. This was also essentially in agreement with the $\text{in vitro}$ lipolytic data from adult rats. These data demonstrate lipolytic differences between depots that are minimal in young rats and which are accentuated with age.     

A procedure for the preferential radioiodination of small amounts of protein in the presence of excess lipids.

The chloramine-T, iodine monochloride, and lactoperoxidase radioiodination procedures were evaluated for their ability to label proteins in the presence of large amounts of reactive lipid. Mouse serum very low density lipoproteins (VLDL) (10% protein, 90% lipid) and detergent-disrupted Friend murine leukemia virus (FLV) (60% protein, 33% lipid) were used as model systems. Based on the distribution of label between protein and lipid, as well as the total amount of label incorporated by each procedure, only a modification of the iodine monochloride procedure preferentially labeled proteins to a high specific activity in both VLDL and FLV. The technique, which is described in detail, is technically simple, rapid, and causes no detectable degradation of proteins.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Novel heme: O2 bonding of possible relevance to oxygen utilizing heme and other proteins

Solid dipyridine hemes which are unreactive toward oxygen lose both pyridine ligands upon heating under vacuum to give a solid which takes up O₂, reversibly, one O₂ per heme. Replacement of ¹⁶O₂ by ¹⁸O₂ reduces only infrared bands near 1660 and 1590 cm^?1, frequencies near the vibrational band for gaseous O₂. No FeO bands are detected. EPR spectra reveal a free radical and ferric iron; Mössbauer, NMR and infrared spectra support an iron(III) oxidation state. Limited molecular weight data indicate a dimer. Possibly two dioxygen molecules are held sandwich fashion between two porphyrins via donor-acceptor interactions, which are facilitated by electron transfer from iron(II) into the porphyrin forming a π-anion. Such O₂ bonding is not found in oxy Hb and Mb or in oxyhemerythrin but may occur with cytochrome $\text{c}$ oxidase and other oxygen utilizing (or producing) heme and other proteins.     

Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis

The activity of 3-hydroxy-3-methylglutaryl CoA reductase in homogenates of the corpora allata of the tobacco hornworm, $\text{Manduca sexta}$, was competitively inhibited by compactin. The K_I for the sodium salt form of compactin was 0.9 nM for the reductase from both male and female corpora allata. In intact female corpora allata juvenile hormone biosynthesis was also inhibited by approximately 50 percent at 10 nM compactin. Following injection with compactin, darkening of the cuticle, an indication of juvenile hormone deficiency, was observed in larvae after ecdysis from third to fourth instar. Hence, compactin shows potential as an inhibitor of insect growth and development.     

The determination of the specific activity of picomolar amounts of long-chain acylcarnitine esters

Measurement of the specific activity of cellular pools of long-chain acylcarnitines is complicated by interference of other labeled cellular lipids, especially phosphatidylcholine and sphingomyelin. To overcome these problems the lipid extract from rabbit aorta labeled with [1-¹⁴C]palmitate was treated with phospholipase C. Upon two-dimensional thin-layer chromatography, the long-chain acylcarnitines could be isolated in an area free of interfering radioactivity. Mobility of long-chain carnitines was inversely proportional to the fatty acid chain length. The amount of long-chain acylcarnitine was quantified from their carnitine content after alkaline hydrolysis using carnitine acetyltransferase.     

Prediction of the secondary and tertiary structure of plastocyanin.

The amino acid sequences of nine plastocyanins were examined using four published methods for the prediction of secondary structure in proteins. The results of the four methods were combined in such a way as to maximize agreement, and the position of alpha helices, beta sheets, and beta turns in plastocyanin was predicted. From this result and other information, such as the position of conserved residues and the requirements for coordination of copper, a preliminary model for the mainchain folding of the molecule was presented.     

ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.     

Immunochemical studies on blood groups: Purification,chemical and immunochemical properties of blood group-active glycoproteins from horse gastric mucosae

Digestion of the gastric mucosae of 10 horses with pepsin or Pronase was followed by phenol/ethanol fractionation. Chemical and immunochemical examination of the fractions showed the mucosae to possess various combinations of A, B and H activities. Most were B-active, three had weak A activity, one had strong H activity and the remainder were weakly H-active; one mucosa possessed neither A, B nor H activity. Digestion with pepsin or Pronase of different portions of the same mucosa yielded products equivalent in serological and most chemical properties. Materials digested by Pronase tended to have less peptide nitrogen than those treated with pepsin. Fractions with the strongest serological activities contained significantly higher amounts of carbohydrate and lesser amounts of peptide nitrogen than those with weak A, B or H activity or with no activity. All mucosae, independent of their A, B or H activity, reacted with concanavalin A. The fractions precipitable by 10% ethanol from 90% phenol reacted most strongly.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.     

A computer program for determining the size of DNA restriction fragments

A computer program has been developed for determining the sizes of DNA restriction fragments from their electrophoretic mobilities. A parabola is fitted to the mobilities of a set of standard fragments of known sizes and the sizes of the unknown fragments are then calculated from the fitted curve. This procedure is shown to yield estimated sizes which are accurate to within a few percentage, as judged by experiments with fragments obtained by digestion of pBR322 with the restriction endonuclease HaeIII. The program, which is written in BASIC, is simple to use and is very much faster than the graphical method that it replaces.     

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Liquid chromatography of pyridine nucleotides and associated compounds and isolation of several analogs of nicotinamide adenine dinucleotide phosphate.

A single-column technique is described for separating the naturally occurring nicotinic acid- and nicotinamide-containing nucleosides and nucleotides. A unique feature of the system is the use of conductivity measurements as a reproducible means of peak identification. The chromatographic system has been used to isolate a number of NADP⁺ analogs and to characterize their enzymatic hydrolysis products. These analogs include the α-anomer of NADP⁺, the nicotinic acid analog of NADP⁺, and a series of NADP⁺ derivatives that contain phosphate in the 3′-position.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

Inhibitors of superoxide dismutases: A cautionary tale

An extensive search resulted in the identification of pamoic acid as an inhibitor of superoxide dismutases. Pamoic acid appeared to rapidly and reversibly inhibit all types of superoxide dismutases and did so in both the cytochrome c reduction and in the dianisidine photooxidation assays, used to measure this activity. It could nevertheless be shown that pamoic acid did not at all inhibit superoxide dismutase but rather diminished the sensitivity of the assays. The mechanism proposed to account for this effect involved oxidation of pamoate, by O₂^?, to yield a pamoate radical which can then reduce cytochrome c or oxidize pyrogallol. Pamoate thus competes with superoxide dismutase for the available O₂^?, without affecting the observable effects of that O₂^? upon cytochrome c or upon pyrogallol. It consequently makes these assays less responsive to superoxide dismutase, while appearing to be without effect in the absence of superoxide dismutase. Several of the predicted consequences of this proposal were affirmed. Other workers, interested in finding inhibitors for superoxide dismutases, are hereby forwarned of this subtle snare.     

Solvent interactions with the active site of the pig heart cytosolic aspartate aminotransferase.

Aqueous solvent interactions with the chromophoric pyridoxal phosphate prosthetic group of aspartate aminotransferase (EC 2.6.1.1) were analyzed quantitatively with ethylene glycol, glycerol, dimethylsulfoxide (DMSO), sucrose, and xylitol as cosolvents. The smaller cosolvents perturb the visible absorption and visible dichroic spectra of the free enzyme, but this solvent perturbation is not observed with the acidic enzymeglutarate complex. Addition of cosolvents caused an increase in the enzyme's affinity for glutarate. This increase in affinity resulted from an increase in the acidic dissociation constant (pK₂) of the enzyme-glutarate complex. The changes in the acidic dissociation constant of the enzyme-glutarate complex, upon addition of cosolvents, correlate well with the changes observed in the pK_a's of carboxylic acids in comparable solvents. Since these solvents have little effect on the pK_a of the enzyme itself, it is concluded that the increase in affinity is due to a specific solvation effect on a carboxyl group of the enzymebound glutarate, rather than resulting from a conformational change in the protein.     

Determination of simultaneous turnover of serotonin,dopamine and norepinephrine in the telencephalon of unrestrained behaving rats

Rats received an injection of tritiated tryptophan and tyrosine via a chronic indwelling jugular catheter, and at 60 and 90 minutes post-injection, were killed by near-freezing. The three biogenic monoamines were separated by ion exchange and thin layer chromatographyand were quantitated with respect to radioactivity and content. The decline in specific activities can be used to calculate rate constants and turnover of serotonin, dopamine and norepinephrine concurrently in unrestrained behaving animals.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.