Creatine kinase isoenzyme profiles after exercise in the rat: sex-linked differences in leakage of CK-MM.

Changes in creatine kinase (CK) activity and CK isoenzyme profiles in plasma after exercise were studied in rats in order to establish the source of the exercise-induced rise in CK activity. Male and female rats ran on a treadmill for 2 h and blood samples, taken before and after exercise, were assayed for total CK, CK isoenzymes and aminoaspartate transaminase (AST) activity. These enzymes were also assayed in homogenates of liver and several muscles. We found that the isoenzyme composition of liver, plasma and muscle did not differ between the sexes. However, the exercise-induced CK and AST responses did differ: CK and AST increased after exercise in males (101% and 15% resp.), but much less in females (47% and 1%). Although the isoenzyme profiles in rest did not differ, significant differences were observed after running: in males CK-MM increased with 678%, but females only showed a 114% increase. In contrast, CK-BB showed a small increase that was about the same for both sexes (males 41%, females 35%). We conclude that both males and females show a small and similar increase in CK-BB activity after exercise, and that a large release of CK-MM from skeletal muscle, observed only in males, accounts for sex-linked differences reported earlier.

Albumin and immunoglobulin-G in the cerebrospinal fluid and the diagnosis of multiple sclerosis.

Multiple sclerosis (MS) remains a diagnosis based mainly on clinical criteria. Definitive diagnostic tests are, despite numerous attempts, not available. However, some laboratory tests like electrophoresis of the cerebrospinal fluid proteins or the determination of immunoglobulin G in cerebrospinal fluid have proved useful in increasing the probability of MS. This paper describes how these laboratory tests have to be interpreted in relation to pathophysiological phenomena and how they correlate with each other. The electrophoresis of CSF proteins and the diagnostic quotients (Ralb and CSF-IgG-index) are valuable aids to the clinician. It especially can substantiate the diagnosis MS. The magnitude of the index cannot be correlated with the clinical stage of MS. The usefulness of these tests in terms of a possible gain of information in the diagnostic process is discussed.

Rapid immunoturbidimetric assay of albumin and immunoglobulin G in serum and cerebrospinal fluid with an automatic discrete analyser.

A method is presented to determine albumin and immunoglobulin G (IgG) in serum and cerebrospinal fluid (CSF) with an automatic discrete analyser. Concentrations of albumin and IgG in serum and CSF are compared with values obtained by radial immunodiffusion (RID). Ratios of albumin in CSF over albumin in serum and the so-called IgG index are calculated. Ratios obtained with the discrete analyser and with the RID are compared and discussed with respect to clinical diagnosis.

Specificity of the phosphatidylcholine exchange protein from bovine liver.

The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver.

The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.

Delipidation of the phosphatidylcholine exchange protein from beef liver by detergents.

1. A method is described to introduce [14C] phosphatidylcholine into the phosphatidylcholine exchange protein from beef liver. The effects of various detergents on this 14C-labelled phospholipid - protein complex are considered. 2. As shown by spectrophotometric and radioactivity analysis of polyacrylamide gels, sodium deoxycholate, Triton X-100, lysophosphatidylcholine and sodium dodecyl sulfate delipidate the exchange protein, while mixed phosphatidylcholine-detergents micelles are formed. 3. Protein delipidated by sodium deoxycholate, Triton X-100 and lysophosphatidylcholine retains its ability to catalyze the transfer of phosphatidylcholine between membranes. The immunological properties are similar to those of native protein as shown by double immunodiffusion in agar against an antiserum gamma-globulin fraction. 4. Sodium dodecyl sulfate and cetyltrimethylammonium bromide interact very strongly with the protein conferring their charge to the complex and destroying the antigenic determinants.