Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

NAD+-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD+ hydrolysis

We previously demonstrated inhibition of Na⁺-dependent ³²P_i transport in canine renal brush-border membranes in association with NAD⁺-induced ADP ribosylation of membrane protein(s) and postulated that NAD⁺ inhibits P_i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD⁺ resulted in release of P_i which was prevented by EDTA. It was proposed that NAD⁺-mediated inhibition of ³²P_i transport might occur through this mechanism. To determine whether NAD⁺ inhibited ³²P_i transport by a mechanism other than or in addition to release of P_i, we compared Na⁺-dependent ³²P_i counterflow in brush-border membrane equilibrated with P_i or with P_i generated from NAD⁺. Release of P_i from NAD⁺ incubated with brush-border membrane was confirmed. The increased uptake of ³²P_i which was demonstrated in brush-border membrane equilibrated with P_i was not measured when intravesicular P_i was generated from a concentration of NAD⁺ which effected ADP-ribosylation of brush border membranes (100 μM NAD⁺). In contrast, increased uptake of ³²P_i was demonstrated when intravesicular P_i was generated from 1 μM NAD⁺ which did not effect ADP ribosylation. Mg²⁺-dependent ADP ribosylation of brush-border membrane incubated with NAD⁺ was demonstrated which persisted during the time interval of ³²P_i uptake measurements. Our findings are compatible with the hypothesis that NAD⁺-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P_i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg²⁺ and decreased ADP ribosylation.     

The postribosomal particle of rabbit liver contains protein-synthesis factors and serum albumin mRNA

As demonstrated by indirect immunoprecipitation and polyacrylamide gel electrophoresis, an 85S particle separated by sucrose density-gradient centrifugation from the postribosomal pellet of rabbit liver, is able to synthesize serum albumin if supplemented with both ribosomal subunits and sources of energy. It is retained on heparin bound to Sepharose 4B, contains translatable mRNA and apparently all protein factors required for translation. This particle may represent a highly organized protein synthesizing machinery, the combination of which with ribosomes results in formation of new protein molecules.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

A high recovery method for concentrating microgram quantities of protein from large volumes of solution

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.