Effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver microsomes: the protein stimulates Ca2+ release

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in the liver microsomes from fed rats was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following addition of 10 mM adenosine triphosphate (ATP). 45Ca2+ uptake was retarded by the presence of regucalcin (1.0-4.0 microM). This retardation was remarkable at 1 min after regucalcin addition, while appreciable retardation was no longer seen at 5 min. Regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was prevented by the presence of calmodulin (5 micrograms/ml). Calmodulin alone (1 and 5 micrograms/ml) caused a significant increase in 45Ca2+ uptake at 3 min after the start of incubation. Also, regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was completely blocked by the presence of a Ca2(+)-trapping agent, oxalate (3 mM). On the other hand, 45Ca2+, which accumulated in microsomes during 5 min after ATP addition, was markedly released by the addition of regucalcin. This release was dose-dependent (0.5-4.0 microM). Guanosine triphosphate (GTP; 10-100 microM) caused a significant release of 45Ca2+ from the microsomes. The presence of regucalcin (2.0 microM) further enhanced the GTP effect. Regucalcin (2.0 microM)-induced release of 45Ca2+ was not blocked by the presence of the protein thiol-protecting agent dithiothreitol (0.1 mM). The presence of oxalate (3 mM) completely blocked the effect of regucalcin on 45Ca2+ release from the microsomes. These results indicate that regucalcin stimulates Ca2+ release from liver microsomes, and that the protein retards the microsomal Ca2+ uptake. The present study suggests that regucalcin can regulate the Ca2+ transport system in rat liver microsomes.     

Activation of hepatic microsomal Ca2+-adenosine triphosphatase by calcium-binding protein regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+-adenosine triphosphatase (ATPase) activity in hepatic microsomes was investigated. Mg2+-ATPase activity was clearly increased by the presence of 50 microM Ca2+. Regucalcin (1.0-4.0 microM) caused a remarkable elevation (about 3-fold) of Ca2+-ATPase activity. Also, Mg2+-ATPase activity was increased (about 1.6-fold) by the presence of regucalcin (2.0 and 4.0 microM). Guanosine-5'-O-(3-thiotriphosphate) (GTPrs; 10(-5) and 10(-4) M) and nicotinamide adenine dinucleotide phosphate oxidized form (NADP+; 10(-5) to 10(-3) M) or reduced form (NADPH; 10(-4) and 10(-3) M) significantly increased Ca2+-ATPase activity. These increases were not enhanced by the presence of regucalcin (2.0 microM). Of various metal ions, a comparatively low concentration of V5+ (10(-5) M) or Cd2+ (10(-6) M) significantly increased Ca2+-ATPase activity, while Hg2+, Zn2+, Cu2+ and Mn2+ did not have such an effect. Regucalcin (2.0 microM) did not enhance the effect of V5+ and Cd2+ on Ca2+-ATPase activity. The present finding, that regucalcin activates hepatic microsomal Ca2+-ATPase, suggests a cell physiological role of regucalcin as an activator in the microsomal Ca2+-pump activity. This action of regucalcin may not be influenced by other regulators.     

Calcium-binding protein regucalcin stimulates the uptake of Ca2+ by rat liver mitochondria

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver mitochondria was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate mitochondrial 45Ca2+ accumulation. 45Ca2+ uptake was stimulated by the presence of regucalcin (1.0 and 2.0 microM). This stimulation was remarkable during 1.0 min after 45Ca2+ addition, while appreciable stimulation was no longer seen at 3 min. Regucalcin (2.0 microM)-induced stimulation of 45Ca2+ uptake was prevented by the presence of ruthenium red (1.0 microM) and lanthanum chloride (0.1 mM). Regucalcin (2.0 microM) did not increase the mitochondrial adenosine triphosphatase (ATPase) activity during 3.0 min after Ca2+ addition. Meanwhile, 45Ca2+, which accumulated in the mitochondria during 5.0 min after 45Ca2+ addition, was not released by the addition of regucalcin. Regucalcin may stimulate Ca2+ uptake in rat liver mitochondria independently of the energy.     

Immunohistochemical demonstration of calcium-binding protein regucalcin in the tissues of rats: the protein localizes in liver and brain

A calcium-binding protein, regucalcin, was isolated from rat liver cytosol. Rabbit-anti-regucalcin antiserum, which was raised against regucalcin conjugated by glutaraldehyde to bovine serum albumin, was applied to glutaraldehyde-fixed whole mounts and subsequently visualized using the peroxidase-antiperoxidase methods. Rat hepatic regucalcin immunoreactivity was most pronounced in the liver and brain of rats, while it was not seen in the duodenum, testicle, spleen, lung and smooth muscle (bladder), and appeared only slightly in the kidney and heart. Control experiments using non-immune sera or adsorbed anti-rat liver regucalcin antiserum showed no staining. The present finding suggests that regucalcin localizes in the liver and brain of rats.     

Effects of Ca2+ and V5+ on glucose-6-phosphatase activity in rat liver microsomes: the Ca2+ effect is reversed by regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on glucose-6-phosphatase in the microsomes of rat liver was investigated. Addition of Ca2+ up to 2.5 microM to the enzyme reaction mixture caused a significant increase of glucose-6-phosphatase activity in hepatic microsomes, while Ni2+, Zn2+, Cd2+, Cu2+, Mn2+ and Co2+ (20 microM) did not have an appreciable effect. Vanadate (V5+) markedly inhibited the enzyme activity; a significant inhibitory effect was seen at 10 microM V5+. The Ca2+-induced increase of glucose-6-phosphatase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcium had no effect on the basal activity of the enzyme. Meanwhile, the inhibitory effect of V5+ (10-100 microM) on glucose-6-phosphatase was not appreciably blocked by the presence of regucalcin (up to 2.0 microM). The present data suggest that hepatic microsomal glucose-6-phosphatase is uniquely regulated by Ca2+ and V5+, of various metals, and that the Ca2+ effect is reversed by regucalcin. The present study supports the view that regucalcin plays an important role as a regulatory protein in liver cell function related to Ca2+.     

Non-respiring rat liver mitochondria do not have a Ca2+/2H+ antiporter

Liver mitochondria take up Ca2+ by the Ca2+ uniporter, whereas at steady state efflux is believed to occur mainly by means of a ruthenium red-insensitive Ca2+/2H+ antiporter. The latter activity was studied in respiration-inhibited mitochondria in the presence of ruthenium red and was measured as Ca2+ uptake following acidification of the matrix by addition of nigericin, which catalyzes K+/H+ exchange. Ca2+ uptake was stimulated by protonophorous uncoupling agents and inhibited by increasing the concentration of ruthenium red. However, the rates were always smaller than those obtained by addition of valinomycin instead of nigericin. This indicates that under these conditions, Ca2+ fluxes are not mediated by a Ca2+/2H+ antiporter but by residual uniporter activity.     

A Ca2+-phospholipid-dependent protein kinase from cottonplant shoots

The Ca²⁺-phospholipid-dependent protein kinase from cottonplant shoots was purified by chromatography on DEAE-Sepharose CL-6B, and then on phenyl-Sepharose CL-4B. According to electrophoresis in PAAG, the enzyme was practically homogeneous and had a molecular mass of 57 kDa. In the presence of Ca²⁺ alone, the enzyme was activated to only a slight degree. Under the combined action of Ca²⁺ and a phospholipid the action of the enzyme rose severalfold. A determination of amino acid specificity showed that the protein kinase isolated was a serine- and threonine-specific protein kinase.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 627071. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 100–105, January–February, 1994.     

Investigating and characterizing the binding activity of the immobilized calmodulin to calmodulin-dependent protein kinase I binding domain with atomic force microscopy

Protein–protein interactions are responsible for many biological processes, and the study of how proteins undergo a conformational change induced by other proteins in the immobilized state can help us to understand a protein’s function and behavior, empower the current knowledge on molecular etiology of disease, as well as the discovery of putative protein targets of therapeutic interest. In this study, a bottom-up approach was utilized to fabricate micro/nanometer-scale protein patterns. One cysteine mutated calmodulin (CaM), as a model protein, was immobilized on thiol-terminated pattern surfaces. Atomic Force Microscopy (AFM) was then employed as a tool to investigate the interactions between CaM and CaM kinase I binding domain, and show that the immobilized CaM retains its activity to interact with its target protein. Our work demonstrate the potential of employing AFM to the research and assay works evolving surface-based protein–protein interactions biosensors, bioelectronics or drug screening.     

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.     

A synthesis of 2-endo-amino-2-exo-hydroxymethylnorbornenes having inhibitory activity against protein kinase C

2-endo-Hexadecylamino-2-exo-hydroxymethylnorbornene (1a) was synthesized from 2-acetamidonorbornene-2-carboxylic acid methyl ester (2) in a good overall yield. 2-endo-Hexadecylamino-2,3-exo-bis(hydroxymethyl)norbornene (1b) was synthesized starting from dimethyl acetamidofumarate based on Diels-Alder strategy. 1a and 1b inhibited protein kinase C at the IC51 values of 2 x 10(-5) and 1 x 10(-5) M, respectively, but not protein kinase A at a concentration of 1 x 10(-3) M. The structure-activity relationships are discussed.     

Chronological protein synthesis in regenerating rat liver

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, ³⁵S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through ³⁵S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5–5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.