Target sizes of human erythrocyte membrane Ca2+-ATPase and Mg2+-ATPase activities in the presence and absence of calmodulin

We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase.     

Identification and characterization of DNA clones encoding group-II glycinin subunits

Summary DNA clones that encode the group-II subunits of soybean glycinin were identified and compared with clones for group-I subunits. The group-I clones hybridize weakly to those from group-II at low stringency, but fail to hybridize with them at moderate or high stringency. The genes for the group-II subunits are contained in 13 and 9 kb EcoRI fragments of genomic DNA in cultivar CX635-1-1-1. These fragments contain genes for subunits A₅A₄B₃ and A₃B₄, respectively. The larger size of mature group-II subunits compared with group-I subunits is correlated with a larger sized mRNA. However, the gross arrangement of introns and exons within the group-II coding regions appears to be the same as for the genes which encode group-I subunits. Messenger RNA for both groups of glycinin subunits appear in the seed at the same developmental interval, and their appearance lags slightly behind that of mRNAs for the a/a subunits of -conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.Abbreviations bp base pairs - kb kilobase pairs - SDS sodium dodecyl sulfate Cooperative research between USDA/ARS and the Indiana Agric. Expt. Station. This work was supported in part by grants from the USDA Competitive Grants Program and the American Soybean Association Research Foundation. This is Journal Paper No. 10,078 from the Purdue Agricultural Experiment Station     

Simplified purification and testing of colloidal gold probes

A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

Topoisomerase I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis.

Topoisomerase I is in situ associated with DNaseI hypersensitive sites located in the promotor and terminator regions of the extrachromosomal rDNA in Tetrahymena thermophila at sites with sequences fitting the motif (sequence in text) Reconstitution experiments with purified topoisomerase I and cloned fragments of rDNA demonstrate that the enzyme exhibits the same binding and cleavage properties on naked DNA. These observations are striking as topoisomerase I previously has been found to exhibit low sequence specificity. The specific binding of the enzyme has an absolute requirement for divalent cations with a preference for Ca2+. The strong binding to the hexadecamer has been characterized by competition experiments, and it has been used to determine the molecular weight of the enzyme.     

Effect of vasoactive intestinal polypeptide (VIP) on steroidogenesis in women

The VIPergic nervous system appears to be the major peptide-containing neuronal component in the female genital tract. Evidence has been put forward that exogenous VIP is able to stimulate progesterone secretion. In the present study the effect of human VIP (900 pmol/kg body weight per h i.v. during 30 min) on steroidogenesis in six female volunteers was investigated. The experiments were performed between the 6th and 14th day of their menstrual cycle, and peripheral venous blood was collected before, during and after infusion of VIP. The concentrations of VIP, oestradiol, progesterone, testosterone, androstenedione (AD), dihydrotestosterone (DHT), dehydroepiandrosterone sulphate (DHAS), sex hormone binding globulin (SHBG) and cortisol were measured. The infusion of VIP was accompanied by a 15% increase (P less than 0.05) in serum oestradiol concentrations, from a mean basal concentration of 0.58 nmol/l. The concentrations of testosterone and DHT also increased significantly. No effect of VIP on progesterone, AD, DHAS, SHBG or cortisol was observed. In the light of the presence of VIP in nerve fibres of the steroid producing tissue, this stimulatory effect of VIP might reflect a direct action on the ovary or the adrenal gland.     

Cat pancreatic eicosapeptide and its biosynthetic intermediate. Conservation of a monobasic processing site.

Pancreatic eicosapeptide is synthesized together with the hormone pancreatic polypeptide in a common precursor in the major endocrine cell type of the duodenal pancreas. This processing has been previously demonstrated in man and in the dog. In the present study the cat pancreatic eicosapeptide and a C-terminally extended form of this were isolated and characterized from acid/ethanol extracts of pancreas by gel filtration and reverse-phase h.p.l.c. The sequence homology in the C-terminal part of the eicosapeptides from different species was shown to continue to the other side of the monobasic cleavage site in the extended intermediate form, whereas the end of the extension differed both in chain length and amino acid sequence. It is concluded that the processing site in the intermediate form of the pancreatic eicosapeptide is an example of a proline-directed monobasic cleavage site that has been conserved during evolution.     

Fluid balance in exercise dehydration and rehydration with different glucose-electrolyte drinks

After exercise dehydration (3% of body weight) the restoration of water and electrolyte balance was followed in 6 male subjects. During a 2 h rest period after exercise, a drink of one of four solutions was given as 9 X 300 ml portions at 15 min intervals: control (C-drink), high potassium (K-drink), high sodium (Na-drink) or high sugar (S-drink). An exercise test (submaximal and supramaximal work) was performed before dehydration and after rehydration. Dehydration reduced plasma volume by 16%, a process reversed on resting even before fluid ingestion began, due to release of water accumulated in the muscles during exercise. After 2 h rehydration, plasma volume was above the initial resting value with all 4 drinks. The final plasma volumes after the Na-drink (+14%) and C-drink (+9%) were significantly higher than after the K- and S-drinks. The Na-drink favoured filling of the extracellular compartment, whereas the K- and S-drinks favoured intracellular rehydration. In spite of the higher than normal plasma volume after rehydration, mean heart rate during the submaximal test was 10 bpm higher after rest and rehydration than in the initial test, and was not different between the drinks. The amount of work which could be performed in the supramaximal test (105% VO2max) was 20% less after exercise dehydration and subsequent rest and rehydration than before. This reduction was similar for all drinks, and may be due to a decreased muscle glycogen content (70% of initial) at the time of the second test.     

Effect of captopril on kidney function in insulin-dependent diabetic patients with nephropathy.

The influence of angiotensin II on kidney function in diabetic nephropathy was assessed by studying the effect of 12 weeks'' monotherapy with captopril (25-50 mg twice a day) in 16 hypertensive insulin dependent diabetic patients with persistent albuminuria. In an initial one week randomised single blind trial of captopril versus placebo, captopril (for nine patients) reduced arterial blood pressure from 148/94 (SD11/6) to 135/88 (8/7) mm Hg (p less than 0.05) and albuminuria from 1549 (range 352-2238) to 1170 (297-2198) micrograms/min (p less than 0.05), while glomerular filtration rate remained stable. No significant changes occurred in seven patients treated with placebo. During the 12 weeks of captopril treatment arterial blood pressure in all patients fell from 147/94 (11/6) to 135/86 (13/7) mm Hg (p less than 0.01), albuminuria fell from 1589 (range 168-2588) to 1075 (35-2647) micrograms/min (p less than 0.01), and glomerular filtration rate fell from 99 (SD19) to 93 (25) ml/min/1.73 m2 (p less than 0.01). The renin-angiotensin system showed suppressed plasma concentrations of angiotensin II and increased concentrations of angiotensin I and renin. The study showed that glomerular filtration rate is not dependent on angiotensin II, that captopril reduces albuminuria, probably by lowering glomerular hypertension, and that captopril represents a valuable new drug for treating hypertension in diabetics dependent on insulin with nephropathy.