Calcium pumping rate

The sodium and calcium pumps can be isolated to near purity and still exhibit most of the biochemical properties of the native pump. Some kinetic properties of these pumps in native membranes are altered or disappear as membrane preparations are purified. For example, when measured in intact membranes, the time-dependencies of phosphorylation and dephosphorylation of the pump catalytic sites exhibit biphasic fast to slow rate transition this characteristic progressively disappears as the membranes are treated with mild detergents. One suggested explanation is that, as the pumps begin to cycle, the catalytic subunits associate into higher oligomers that may permit more efficient transfer of the energy from ATP into the ion transport process [29, 30], Some structural evidence indicates that Na,K pumps exist in cell membranes as multimers of (a 3)2 [31]. 

The transport of calcium into the mitochondrion can lower external Ca2+ to levels of 1 to 0.1 jumole/1. This has, therefore, been interpreted as a basic mechanism in maintaining intracellular calcium at these levels. Only about 3 % of the calcium which passively diffuses into the cell is expelled by a calcium pump into the plasma membrane, whereas the remaining 97 % is sequestered into the mitochondria. This occurs because both processes have similar rate constants, but the total mitochondrial surface is some 30 times larger than that of the plasma membrane. This argument presupposes, however, that the calcium which enters the cell is equally available to both sets of membranes. 

SERCA pumps in cultured COS cells. Their functional properties were studied using isolated microsomes. The initial in vitro studies reported that SERCA1 and SERCA2a isoforms shared similar Ca2+ affinity and velocity of Ca2+ uptake (Vmax). Subsequently, a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform (5.0- versus 2.6-fold increase in calcium uptake rate) (Sumbilla et al 1999). 

This obvious dependence on extracellular calcium is somewhat unexpected because (1) the sustained enhancement of calcium influx rate is adequately balanced by an increase in calcium efflux rate so that (2) the calcium concentration in the bulk cytosol is maintained near the basal value. This apparent paradox may be resolved by a model [54] which postulates that during the sustained phase of cellular response the high rate of calcium cycling across the plasma membrane raises the calcium concentration in a region just below the plasma membrane, often called the submembrane domain (see Rasmussen and Barrett, Chapter 4). Because the elevated calcium level in this domain is not conducted into the bulk cytosol, it cannot activate calcium-dependent response elements in the cytosol. Rather it regulates the activity of calcium-sensitive, plasma membrane-associated enzymes such as the calcium pump and PKC, the previously described phospholipid-dependent, calcium-activated protein kinase. 

The sarcoplasmic reticulum vesicles start to release calcium passively when the activity of the calcium pump is blocked, either by inhibition of the transport enzyme or by depletion of energy-yielding substrates [45]. The rate of passive calcium efflux from vesicles loaded in the absence of calcium-precipitating agents, is approx. 20... 

From a practical point of view the major difference between the two approaches to enzyme kinetics, steady state and transient rate measurements, is in the concentrations of enzyme used. Steady state experiments are carried out with catalytic amounts of enzyme at concentrations negligible compared to those of the substrates or products. The rationale of transient kinetic experiments, discussed in section 5.1, will be seen to rest on the observation of complexes of enzymes with substrates and products. The importance of the direct observation and characterization of reaction intermediates for an understanding of mechanisms will be illustrated in that section. This requires enzyme concentrations sufficiently high for detection of intermediates by spectroscopic or other physical monitors. There are a number of interesting systems in vivo with enzyme and substrates at comparable concentrations and the potential kinetic consequences of such situations will be discussed in sections 5.2 and 5.3. Jencks (1989) comments in connection with a review of the transient kinetic behaviour and mechanism of one such system, the calcium pump of the sarcoplasmic reticulum, that steady state kinetics could make no contribution to an understanding of its ATPase linked reaction. The same can be said of the mechanism of myosin-ATPase, which has been elucidated in detail by transient kinetic studies (see section 5.1). 

Ethyl phenylethylmalonate. In a dry 500 ml. round-bottomed flask, fitted with a reflux condenser and guard tube, prepare a solution of sodium ethoxide from 7 0 g. of clean sodium and 150 ml. of super dry ethyl alcohol in the usual manner add 1 5 ml. of pure ethyl acetate (dried over anhydrous calcium sulphate) to the solution at 60° and maintain this temperature for 30 minutes. Meanwhile equip a 1 litre threenecked flask with a dropping funnel, a mercury-sealed mechanical stirrer and a double surface reflux condenser the apparatus must be perfectly dry and guard tubes should be inserted in the funnel and condenser respectively. Place a mixture of 74 g. of ethyl phenylmalonate and 60 g. of ethyl iodide in the flask. Heat the apparatus in a bath at 80° and add the sodium ethoxide solution, with stirring, at such a rate that a drop of the reaction mixture when mixed with a drop of phenolphthalein indieator is never more than faintly pink. The addition occupies 2-2 -5 hoius continue the stirring for a fiuther 1 hour at 80°. Allow the flask to cool, equip it for distillation under reduced pressure (water pump) and distil off the alcohol. Add 100 ml. of water to the residue in the flask and extract the ester with three 100 ml. portions of benzene. Dry the combined extracts with anhydrous magnesium sulphate, distil off the benzene at atmospheric pressure and the residue under diminished pressure. C ollect the ethyl phenylethylmalonate at 159-160°/8 mm. The yield is 72 g. 

Fast-twitch muscle fibers develop tension two to three times faster than slow-twitch muscle fibers because of more rapid splitting of ATP by myosin ATPase. This enables the myosin crossbridges to cycle more rapidly Another factor influencing the speed of contraction involves the rate of removal of calcium from the cytoplasm. Muscle fibers remove Ca++ ions by pumping them back into the sarcoplasmic reticulum. Fast-twitch muscle fibers remove Ca++ ions more rapidly than slow-twitch muscle fibers, resulting in quicker twitches that are useful in fast precise movements. The contractions generated in slow-twitch muscle fibers may last up to 10 times longer than those of fast-twitch muscle fibers therefore, these twitches are useful in sustained, more powerful movements. 

The functions of the calcium-storage capacity of the ER are at least threefold the association of Ca2+ with Ca2+-binding proteins in the ER is part of a chaperone function that is essential for normal protein synthesis the rapid rate of Ca2+ uptake by endoplasmic pumps provides shortterm cytoplasmic Ca2+ buffering that resists untoward and transient changes in [Ca2+] and, finally, many signaling pathways employ elevated [Ca2+] to activate physiological processes. Extensive Ca2+ release from ER is coupled to activation of Ca2+ entry across the plasma membrane, a process known as capacitative calcium entry, which is discussed below. 

In sum, the natural tendency will be for sodium, calcium, and chloride ions to flow into the neuron and for potassium ions to flow out, and in so doing to reduce the membrane potential to zero. In reality, this is not so easy. The plasma membrane of the neuron is not very permeable to these ions. If it were, it would be impossible to sustain concentration gradients across it. The rate of passive diffusion of these ions across this membrane is very slow, though not zero, and different for each ion. So how do ions get across the neuronal plasma membrane rapidly There are two ways gated channels and active transport by pumps. 

The sarcoplasmic transport system can be fueled in addition to ATP by a great number of phosphate compounds which differ considerably in their chemical nature. Not only the natural nucleoside triphosphates126 but also para-nitrophenylphosphate,27 acetyl phosphate128 or carbarmyl phosphate129 can drive calcium transport. While there are considerable differences between the rates with which calcium transport proceeds with the different substrates, they are all used with the same coupling ratio of two and the pump can establish similar maximal concentration ratios (Fig. 7)... 

---