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Mg2+- and Ca2+-dependent ATPase activity, fructose and divalent cation concentrations were determined in seminal plasmas from men with oligozoospermic ejaculates and from those with normal sperm count. The mean activity of the Mg2+- and Ca2+-stimulated ATPase from the former was 59 nmol . 0.1 ml-1 . This is significantly (p less than 0.005) lower than for men with a normal sperm count (corresponding figure is 87 nmol . 0.1m1-1 . min-1). As regards the concentrations of Mg2+, Ca2+ and Zn2+ in the seminal plasmas from these two groups there was no significant difference. The same was also valid for the fructose concentrations. The Mg2+- and Ca2+-stimulated ATPase activity as well as fructose and divalent cation concentrations were also determined in seminal plasmas from men before and after vasectomy. After vasectomy, only the ejaculates devoid of spermatozoa were included in the comparative studies. No significant difference was observed between the seminal plasmas from men before and after vasectomy. These findings support the view that the Mg2+- and Ca2+-stimulated ATPase system does not derive from the spermatozoa. Possible explanations for the significantly lowered Mg2+- and Ca2+-dependent ATPase activity from patients with oligozoospermia are given.

Paracorporeal rat hearts were supplied with blood derived from the abdominal aorta of a supporting rat. The circulatory stability and working capacity of the supporting animal was analyzed in the experimental situation in terms of PO2, SO2, PCO2, HCO3, pH and electrolytes, all of which were within the normal range before and during a 60 min period of paracorporeal perfusion. For evaluation of ischemic damage in this model studies were made on three groups of excised hearts. They were subjected to 10, 15 or 20 min of complete global ischemia at 37 degrees C (ambient temperature) and reperfused for 30 min, including ECG, observations of contractility and an analysis of creatine kinase efflux in the coronary effluent. The results showed good reproducibility and the data were in accordance with reports from similar studies on Langendorff preparations. The model, which is easily set up, inexpensive and based upon pulsatile blood perfusion, should be more physiologic than the conventional Langendorff preparation.

Creatine phosphate is a precursor molecule for ATP synthesis, even under ischemic conditions. We investigated its functional and metabolic effects when added to cardioplegic solution. Rat hearts were subjected to normothermic ischemia for 15 or 30 min and then freeze-clamped. During ischemia there was gradual reduction of high-energy phosphates, but the hearts with creatine phosphate supplement showed higher myocardial content of ATP and of the creatine compound. Other hearts, subjected to 20 min of ischemia, were reperfused with blood for 40 min. Creatine phosphate supplementation resulted in better left ventricular isovolumic work during spontaneous activity, but in paced activity (400 beats/min) no significant differences were seen. After reperfusion, supplemented hearts showed a tendency to higher levels of ATP and creatine phosphate. In all three groups the hearts with cardioplegic supplement had significantly increased myocardial content of pyruvate without proportional lactate increase. The results indicate that creatine phosphate may be an effective constituent in cardioplegic solution.

The effect of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP) administered during ischemia was investigated, using the paracorporeal rat heart model. During 15 min of global ischemia the hearts were perfused twice with PEP and ATP (supplemented) or with NaCl only (non-supplemented). In hearts that were freeze-clamped after the ischemic period (group A), the myocardial content of high-energy phosphates showed only minor differences between hearts with and without supplemention. In the supplemented hearts there was increased myocardial content of pyruvate and, to some extent, of lactate, indicating that PEP was metabolized to pyruvate and partly to lactate. In groups B and C the hearts were reperfused for 40 min before freeze-clamping. The non-supplemented hearts showed higher energy content and better left ventricular performance and, concomitantly, the creatine kinase isoenzyme MB (CK-MB) efflux was less than for the supplemented hearts. A clear inverse relationship between left ventricular performance and CK-MB efflux indicated that CK-MB served as a marker of cell integrity. These results contrasted with previus finding of an unequivocally positive effect from PEP and ATP administration. Possible explanations are discussed.

Semen was collected under standardized conditions from men who were divided into three groups: a control group from normal spermiograms, men with abnormal spermiograms, and azoospermic men following vasectomy. Ultracentrifuged seminal plasma was analyzed for uridine, xanthine, urate, and the pelleted material for Mg2+- and Ca2+-dependent ATPase and protein. No significant intergroup differences were noted except the significant elevation of xanthine of vasectomized men. Uridine that occurs in high concentration in seminal plasma displayed a positive correlation to percentage motile sperms from the 26 men with normal and abnormal spermiograms. It was concluded that an optimal secretory function of uridine may parallel the increasing percentage of sperm with a better quality of motility. A linear relationship between ATPase activity and sperm penetration ability existed only when taken into consideration those 13 specimens with lowest enzyme activity.

Seminal plasma from 22 men attending an infertility clinic was subjected to preparative ultracentrifugation for 2 h at 105,000 g. The pelleted material as well as the supernatant thus obtained were investigated with regard to prostasome membrane-linked enzyme activities in relation to other semen parameters. The mean activity of Zn2+-dependent adenosine triphosphatase in the sedimented prostasome fraction was 1.45 +/- 1.02 mumol (range 0.29-4.79) orthophosphate released per milligram protein and 20 min, while the corresponding figures for the supernatant were 0.56 +/- 0.30 (range 0.12-1.29). Hence, 72% of the specific activity was sedimented, and 28% remained in the supernatant. The same pattern was recognized with regard to the other two enzymes investigated, although they displayed individual characteristics with regard to distribution after ultracentrifugation. The pelleted prostasome-linked mean aminopeptidase activity was 0.39 U/mg protein (81.9%), with only 0.087 U (18.1%) remaining in the supernatant. The corresponding figures for gamma-glutamyltransferase were 7.89 (60.4%) and 5.17 (39.6%) mu kat/g protein, respectively. The different enzyme activities in the prostasome fraction and supernatant, respectively, were interrelated to each other and correlated significantly with r values between 0.73 and 0.93 (p less than 0.001). It was concluded that a minor fraction of prostasomes remained in the supernatant after ultracentrifugation. A relationship existed between prostasomes and semen volume revealing a rather consistent pattern in that small volumes favoured the presence of comparatively more prostasomes in the supernatant and less prostasomes in the pelleted fraction than large volumes. In addition, the sperm concentration seemed to be another determinant of the distribution of prostasomes in seminal plasma on subsequent ultracentrifugation.

In order to describe subclinical brain injury in conjunction with cardiac operations 94 patients were prospectively studied with three brain injury assessment methods: CSF analyses 24 hours after bypass, psychometry, and computed tomography of the brain. Adenylate kinase (AK), a marker of ischemic brain cell injury, was measured in cerebrospinal fluid (CSF) and in serum. In 13% of the patients, a considerable increase in CSF-AK was seen, in 46% there was a moderate increase, and in 41 % no or trivial increase. Psychometry measured as change between preoperative and postoperative scores in a test battery (SS3) revealed a moderate decrease in intellectual function after operation. There was a significant inverse correlation between CSF-AK and SS3 (r = −0.46, p < 0.001, r2 = 0.21, n = 71). Computed tomography (CT) of the brain was performed preoperatively and postoperatively in 54 patients. Two of these had cerebral infarctions visible on the CT, despite an essentially normal postoperative state. There was no correlation between indices of brain injury and patient diagnosis and length of perfusion. It is concluded that subclinical brain injury is often seen after cardiac operations. Most often the injury appears trivial and/or reversible, but in a minority of cases there is evidence that the brain injury is irreversible. Factor analysis favors the view that the microembolism theory might no longer be a valid concept in modern cardiopulmonary bypass (CPB). Instead, circumstances in the operative field seem more likely to be important causative factors. This interpretation calls for new principles in the search for an improved cerebral protection during cardiac operations.

Blastocysts recovered from mice in a state of delay of implantation were incubated for 10 h with either alpha-aminoisobuturic acid (AIB) or 2,4-diaminobuturic acid (DAB), two non-metabolizable amino acids. The incubation medium was composed so as to maintain growth arrest of the inactive, delayed blastocysts in vitro. The blastocysts were then transferred to a complete outgrowth medium without the two non-metabolizable amino acids to test the capacity for trophoblast outgrowth. AIB, which displays saturation kinetics, was harmless to the blastocysts even at a high concentration, while DAB at a low concentration irreversibly damaged the trophoblast cells and prevented outgrowth, probably due to nonsaturation kinetics resulting in a high intracellular accumulation. The harmful effect of DAB could be abolished by concomitant incubation with L-alanine and L-methionine, which compete with DAB for the same transport system, while the D-forms of the same amino acids had little or no effect. The results suggest the presence of transport System A in mouse blastocysts growth arrested in vitro, indicating an operative carrier mechanism already during delay of implantation.

A pronounced deficiency of Mg2+ and Ca2+-dependent ATPase and of secretory granules and vesicles was demonstrated in the seminal plasma of a patient with infertility problems (person A) and with a lowered serum testosterone level. Both total ejaculates and different fractions of split-ejaculates were examined on repeated occasions. Divalent cations, fructose and protein were also determined in most of the samples. The low activity of the Mg2+ and Ca2+-dependent ATPase and the ultrastructure of the split-ejaculates of the seminal plasma of person A contrasted sharply against the high activity of the Mg2+ and Ca2+-dependent ATPase and the ultrastructure of the split-ejaculates of the seminal plasma of another individual (person B). The latter displayed a normal Mg2+ and Ca2+-dependent ATPase activity as well as an ordinary representation of the secretory granules and vesicles. The findings on the ATPase system in the seminal plasma of person A together with the lowered levels of divalent cations in the order Ca2+ less than Mg2+ less than Zn2+ are proposed to reflect a defective functioning of the prostate gland in person A.

Acid phosphatase in serum was measured in 116 patients with prostatic disease, benign in 59 and malignant in 57 cases. Comparisons were made between radioimmunoassay (RIA) and an enzymatic method. The correlation coefficient between the respective values was 0.96 in patients with untreated prostatic cancer, indicating that no significant difference between results with the two methods was to be expected. The correlation coefficient between RIA values and cancer stage was 0.48, and between catalytic activity and cancer stage it was 0.50. The validity of the two methods consequently was equal. RIA, however, was the more sensitive method, giving elevated values in 10 of 11 patients with untreated stage III or stage IV prostatic cancer, as compared with only 4 of the same 11 in the enzymatic assay. This seeming paradox most probably was attributable to differing intrinsic properties of the methods when the upper limits of normal range were established. Neither RIA nor enzymatic analysis discriminated early prostatic cancer (stages I and II) from benign lesions.

A Mg2+- and Ca2+-dependent ATPase at the outer surface of human peripheral lymphocytes and hematopoietic cell lines has been studied. The enzyme activity varied about 60 fold between the extremes of the cell lines tested. There was no simple relationship between surface-ATPase activity and cell surface area. Nor was the presence of villi or any other morphological characteristic at the cell surface decisive for the degree of surface-ATPase activity. A correlation was, however, found between the ATPase activity and a phenomenon probably involving a contractile process namely immunoglobulin secretion, but it was not possible to establish an interdependence of these cell characteristics.

The myocardial contents of pyruvate, lactate and orthophosphate after ischemia were investigated in a paracorporeal rat heart model under different conditions. The arterial blood was supplemented with phosphoenolpyruvate (PEP) and adenosine triphosphate early during reperfusion of excised hearts subjected to 15 min of complete global ischemia at 37 degrees C. The increase in myocardial pyruvate was significant after 4 min of reperfusion compared with the content in non-supplemented hearts subjected to the same ischemic trauma. Such dynamic changes were not observed for lactate and orthophosphate under corresponding conditions. The distinct increase in myocardial pyruvate content on supplementation with PEP and adenosine triphosphate was most probably due to an adenosine triphosphate-mediated PEP translocation into myocardial cells, with rapid metabolization of translocated PEP into adenosine triphosphate (in the presence of cellular adenosine diphosphate) and pyruvate. The pyruvate and lactate relationship varied, depending on the postischemic conditions. The postischemic myocardial orthophosphate content was stable, with only minor fluctuations. This possibility to supply the postischemic myocardium with substrate for immediate intracellular energy production is of clinical interest and merits further studies.

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased the the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients. The amount of Mg(2+)-and Ca(+)-dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg2+- and Ca2+-dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent. The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.

The incidence and extent of cerebral damage following open-heart surgery were prospectively investigated in 103 patients, using clinical assessment, psychometry, adenylate kinase analysis in cerebrospinal fluid (CSF-AK) and computed tomography (CT) of the brain. The surgical mortality was 1.9%. Clinically there was obvious cerebral dysfunction in four cases, subtle evidence of brain damage (mainly undue fatigue) in 16 and no evidence in 81 cases. In the 16 patients the mean CSF-AK was substantially increased (0.122 U/l) and the psychometric performance distinctly impaired (-12 points) postoperatively; in the 81 patients the figures were 0.55 U/l and -3.4. Psychometrically, 60% of the patients showed cerebral dysfunction, which was pronounced in 16%. CSF-AK analysis indicated cerebral damage as absent or trival in 45%, moderate in 33% and marked in 22%. CT revealed postoperative cerebral infarction in two cases. Results from the various methods showed reasonable correlation, but also considerable overlap. Open-heart surgery thus can cause brain damage additional to that neurologically discernible. Fatigue is an important sign in this context. In research on postoperative brain damage, the relative insensitivity of routine neurologic investigation calls for supplementary, refined methods.

Adenylate kinase activity and lactate concentration were measured in the cerebrospinal fluid (CSF) of 5 patients with bacterial meningitis, of 4 patients with probable bacterial meningitis, and of 18 patients with serous meningitis. Furthermore, for comparison measurements were also performed in CSF of 27 patients with meningism. Concomitantly glutathione was measured in CSF in most of the patients. Significantly higher values of these 3 parameters were found in the CSF of patients with bacterial and probable bacterial meningitis compared with those having serous meningitis and meningism. Adenylate kinase activity and lactate concentration in patients with serous meningitis were significantly higher than in those with meningism. All patients with a clinical diagnosis of meningitis studied so far also displayed an adenylate kinase activity in their CSF. The determination of adenylate kinase, lactate and glutathione levels in CSF might be a useful aid for the diagnosis not only of meningitis but also for the discrimination between bacterial and serous meningitis.

Adenylate kinase activity was found in 32 of 34 samples of cerebrospinal fluid (CSF) from 21 patients with stroke and seven patients with global cerebral ischaemia (GCI). The light absorbance values of the spectrum 400-650 nm revealed the scanty occurrence of haemoglobin products in the CSF in some patients. No correlation was found between the absorbance values at 415 nm, reflecting oxyhaemoglobin, and the adenylate kinase activities. Thus, a main contribution to the adenylate kinase activity in CSF by leakage of this enzyme from erythrocytes could be ruled out. Instead increased leakiness of the brain cells, having an impaired metabolism due to insufficient supply of oxygen and glucose, was the most plausible cause of the findings. The quotient between the adenylate kinase activity and the light absorbance at 415 nm seemed to reflect the extent of ischaemically deranged brain tissue in the GCI patients, while the CSF-lactate values were not correlated to the clinical outcome. Glutathione, an intracellular tripeptide, was more often found in the CSF from GCI patients than from stroke patients.

Probes for measuring energetic gradients across biological membranes have generally been selected for their chemical inertness with respect to the membrane. For example, the distribution of the chloride ion or of the dibenzyldimethylammonium ion across the red blood cell membrane may serve to report the transmembrane potential gradient if their movements respond to that force and to no other; 5,5-dimethyl-2,4-oxazolidinedione (DMO) will report the cytoplasmic pH only if its movement responds only to the pH gradient and to no other gradient. The same requirements apply to ion-ionophore combinations that are used for similar purposes. If any of these test substances were to encounter specific uphill transport in traversing the membrane, an invalid measurement of the desired gradient would result. Probes of this type, of course, provide only a statistical measurement of the energetic gradient, measured all the way across the membrane and averaged for its total area. For the sake of clarity, we deliberately sidestep the possibility that we could obtain information on local gradients by avoiding any energytransferring reactivity between the probe and components of the membrane. We will develop here the possibility of using quite another type of probe, one whose crossing of the membrane occurs only through the chemical binding and release events implicit in active transport.'. 2 Such probes should be hydrophilic solutes, often zwitterions, sometimes with a net charge, so that their passage by diffusion through the lipoid membrane substance is negligible in relation to their passage by specific transport. Their molecular structure must combine two sets of features: (1) the structural features necessary to keep transport restricted to the given system, which may or may not supply a vectorial force in one or the other direction; and (2) a structural feature that causes a response to a membrane gradient. Control substrates that lack this feature will then provide the reference standard, by comparison with which the response is recognized. Such test substances we will call gradient-sensing substrates for transport. Application of a membrane probe that combines these features would invite only confusion if the membrane also permitted their passive permeation. But when migration is limited to the pathway described by the process of active transport, then we may achieve probes that sense gradients specific to the interval between successive topographic points on that pathway. Rather than obtaining another

A transfer of (32P)-labeled phosphoryl groups has been demonstrated to occur between prelabeled Ehrlich cells (donor cells) and glioma cells (acceptor cells). The Ehrlich cells were generally prelabeled in vivo by an intraabdominal injection of (32P)-orthophosphate 24 hours prior to scarifice of the tumor-bearing mice. In some experiments two injections of (14C)-glucose were given to the animals in addition to the injection of (32P)-orthophosphate prior to sacrifice. The prelabeled Ehrlich cells were mixed with the glioma cells, that were still attached to the culture plates, and incubation took place in vitro. Labeled (32P)-phosphoryl (14C)-serine was isolated from the glioma cells as well as from the Ehrlich cells. The 32P/14C-ratio of (32P)-phosphoryl (14C)-serine of the glioma cells (acceptor cells) was significantly higher than found for the Ehrlich cells (donor cells). This was indicative of an interactive mechanism between the cells partly due to binding of membrane fragments from the Ehrlich cells on to the surface of the glioma cells and partly due to a phosphoryl group transfer between the two types of cells. Interaction experiments with a plasma membrane fraction prepared from the prelabeled Ehrlich cells vis a vis the glioma cells clearly showed that the phosphoryl group transfer mechanism between the cells was dominating and only a minor part of the interaction was due to adhesion. This transphosphorylation reaction represents a new type of interaction between cells. It was not influenced by either adenosine 3', 5'-monophosphate or adenosine 5'-diphosphate.

Seminal plasma constituents were separated on Sephadex G200 gel columns. The column eluate was analysed with regard to protein content, ATPase activity and promotive activity on sperm progressive motility. Two different chromatographic fractions were also subjected to electron microscopy after sedimentation by preparative ultracentrifugation. A maximum promotive value on sperm progressive motility coincided with a maximum ATPase activity value in a single peak from seminal plasma eluted first on the column and containing less protein than the other peaks appearing later in the chromatogram. This first peak was the only one containing ATPase activity and membrane-surrounded organelles named prostasomes. Other peaks, rich in protein but lacking ATPase and prostasomes, displayed a moderate and rather irregular pattern in reference to promotive activity on sperm progressive motility. Evidence is given that the positive effect by prostasomes is specific on sperm progressive motility. Hence, procedures aiming at a change of membrane integrity of the prostasomes resulted in diminished effects on sperm progressive motility. This could be explained by a probable dissipation of the electrochemical gradient of calcium ions.

The surface morphology of membrane fractions of human erythrocytes isolated by gradient centrifugation with a colloidal silica solution (Ludox) differed from the structure of control membranes isolated without the silica solution. The altered surface had a wrinkled appearance with a granular texture, compared with the smooth appearance of the controls. Thus the colloidal silica solution changed the ultrastructure of erythrocyte membranes. Possible mechanisms for the influence of the silica particles are suggested: secondary chemical bonds, e.g. hydrogen bonds, might well have been established between the silica particles and components of the erythrocyte membrane like spectrin of the inner surface of the membrane. Less probable is an attachment to positive sites of the outer side of the membranes as well as any association to the pitlike infoldings of the erythrocyte surface.

The activities of fucosyltransferase, galactosyltransferase, and sialyltransferase were estimated in ultracentrifuged seminal plasma samples from normospermic and oligospermic men. There was no significant difference with regard to these activities between the two groups. Hence, fucosyltransferase activity was high in both normospermic (mean value: 190.2 nmol/l) and oligospermic men (mean value: 157.2 nmol/l) and exceeded that of blood plasma (mean value: 7.6 nmol/l) by 20-25 times. On the contrary, the activity ratio between seminal plasma and blood plasma of galactosyltransferase and sialyltransferase was low and fairly close to 1. A significant correlation was noted between seminal plasma galactosyltransferase activity and semen volume only in normospermic men. Significant inverse correlations in oligospermic men were observed between seminal plasma sialyltransferase activity and sperm concentration (and total sperm count) as well as between galactosyltransferase and fucosyltransferase activities.

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.

The in vivo transport of 14C-AIB (14C-alpha-aminoisobuturic acid) into mouse blastocysts was studied during activation for implantation. Mice, kept in experimentally delayed implantation, were given estrogen to induce implantation and then injections of 14C-AIB i.v. at 0, 4 and 8 h after the estrogen. After in vivo incubation times for 1/3 or 4 h with the labeled amino acid the blastocysts were flushed out of the uterus and collected. A distinct uptake of 14C-AIB occurred in the blastocysts 8 h (the highest uptake) and 12 h after the induction, provided that the in vivo incubation time was 4 h. At these times the blastocysts are lying free in the uterine lumen and consequently there is a transport of 14C-AIB from the epithelium via the uterine secretion into the blastocysts. This uptake indicates that amino acids transported by the system A are important nutrients during early activation. The uptake and retention of 14C-AIB in the uterus was tested at 4 and 8 h after the induction of implantation. The highest uptake was observed when the labeled amino acid was given at 8 h while the longest retention time occurred when 14C-AIB was given at 4 h. Since the transport ratio between the blastocysts and the uterine tissue is not maintained constant it is concluded that the metabolic rates for 14C-AIB transport are different for the uterus and the blastocysts. The AIB transport into uterine tissue preceeds that into the blastocysts. The AIB transport into the blastocysts is maintained as long as they have a negative surface charge.

A stimulation by zinc ions of the hydrolysis of ATP by prostasomes prepared from human semen has been observed. Stimulation was maximal at a Zn2+/ATP stoichiometry of 0.5/l, and increasing this ratio resulted in a gradual decrease in ATPase activity. The pH optimum was 6.0. The apparent Km for Zn2+-dependent ATPase was 0.43 mmol/l and apparent Vmax 5.60 mumol/mg protein/20 min. Other divalent cations could replace Zn2+ as cofactor more or less effectively in the order Mn2+ greater than Cd2+ greater than Ba2+ greater than Sr2+. Potassium ions produced a further activation of the Zn2+-dependent ATPase system by about 10%. Such a stimulation was also attained to some extent by other monovalent cations as Rb+, NH4+, Li+ and to a lesser extent by Cs+. Orthovanadate in the concentration interval 5-1,000 mumol/l was inhibitory of the Zn2+-dependent ATPase system in a dose-dependent fashion. An aminopeptidase activity was also linked to the prostasomes. This enzyme activity was dramatically inhibited by 2 mmol/l orthophenantroline. A reactivation of the orthophenantroline-inhibited aminopeptidase activity was possible by adding Zn2+ to the reaction mixture. Hence, prostasomes contained ATPase as well as aminopeptidase activities both of which being dependent upon Zn2+. These two activities did not seem to be expressions of an ATP-dependent protease activity associated with prostasomes.

An endogenous protein kinase at the surface of Ehrlich cells has been studied. Using exogenous (gamma32P)ATP as a phosphoryl group donator, a transfer was demonstrated into endogenous acceptor protein(s) as well as to exogenous phosvitin. Seryl- and threonyl-residues isolated from the endogenous and exogenous acceptor protein were found to be labeled. The ratio between the labeled phosphorylserine and phosphorylthreonine was about 3.5:1 for both the endogenous acceptor of the intact cells and the exogenous acceptor. In similar experiments with a membrane preparation from Ehrlich cells, this ratio increased to about 7:1 provided the exogenous acceptor protein was absent. The results were independent of whether 1 X 10(-5) M dibutyryl cyclic AMP was used or not with intact cells and a membrane fraction mainly consisting of vesicles. Whether the regulatory subunit of the membrane-associated protein kinase was in cis- or trans-disposition to the catalytic subunit no binding and dependence of the cyclic nucleotide was observed. Since the purified membrane fraction was considered free from endogenous cyclic AMP, it was concluded that the membrane-associated protein kinase of Ehrlich cells is not dependent on cyclic AMP. The critical role of arginine for the cyclic AMP dependence of the serine-containing residue in the catalytic subunit is discussed.

An intact organelle, the prostasome, is secreted by the acinar epithelial cell of the human prostate gland. The ultrastructural location of the prostasome is within membrane-bound storage vesicles in the epithelial cells. Prostasomes are delivered into the glandular lumen by an exocytotic event, which is preceded by fusion of adjacent membranes belonging to the storage vesicle and the epithelial cell. Alternatively, the storage vesicle can be translocated in toto from the cell interior into the acinar lumen through the plasma membrane. This latter event has been designated diacytosis. Both phenomena seem to occur with approximately equal frequency in the human prostate gland. An ATPase system that is Mg2+ and Ca2+-dependent is firmly linked to the membranes encasing the prostasomes. The ATPase system may be the molecular basis for vectorial transport of calcium into these organelles. Also a protein kinase activity is located in the membranes. An increase in membrane thickness was observed on phosphorylation. The physiologic function of the prostasomes is not known. They may be important for promoting forward motility of spermatozoa.

The content of cyclic nucleotides (cAMP, cGMP) and Mg2+, Zn2+,Na+ and K+ was determined in human erythrocytes and after beta-adrenergic blockade in healthy individuals. The figures for cAMP and cGMP were 4.20 and 3.36 nmoles/ l intracellular volume, respectively. Only slight effects on the cyclic nucleotide content as well as on cation content were seen upon beta-blockade in the erythrocytes. It is concluded that the single determination of red cell cAMP is not a sufficient indicator of beta-adrenergic blockade. The determination of cAMP in combination with cGMP, Zn2+ and Na+ might reflect a beta-adrenergic blockade.

Decreased 45calcium uptake was observed in red cells of 20 patients with essential hypertension. Equilibration of extracellular 45calcium with intracellular calcium was not achieved within 60 min in red cells of either hypertensive patients or control subjects. By introducing the ionophore A23187, equilibrium conditions were attained for red cells of both categories of individuals. Still the discrepancy in 45calcium uptake was preserved between them. These results support the view that red cells of hypertensive patients have an altered membrane permeability to calcium, possibly reflecting also a greater exchangeable pool of cytosolic free calcium.

A simple method for objective determination of sperm progressive motility is described. Comparisons were made between results with this method and subjective grading of motility. Although congruence between the two methods was demonstrated, the subjective evaluation had obvious disadvantages. In suspensions of NaCl-washed spermatozoa, seminal plasma was required for progressive motility. Various isotonic salt solutions without seminal plasma were ineffective in this respect. Washed spermatozoa, however, were always motile in a medium consisting of pellet II, obtained from ultracentrifugation of human seminal plasma, thoroughly mixed with isotonic NaCl solution. The progressive sperm motility was two to three times as great in that medium as in seminal plasma mainly devoid of organelles. Magnesium, calcium and zinc ions, in the presence of seminal plasma, could exert stimulatory and inhibitory effects on sperm progressive motility, depending on the concentration of each divalent cation. Magnesium gave the greatest stimulatory and zinc the greatest inhibitory effect.

Supplementation with phosphoenolpyruvate (PEP) and ATP was previously found to enhance the protective effect of potassium cardioplegia on rat hearts subjected to extensive ischemic trauma (30 min at 37 degrees C) in the paracorporeal rat heart model. In the present experiments, the ischemia time was reduced to 20 min (37 degrees C). Ventricular work after ischemia was best in control rats with potassium cardioplegia only. Supplementing the cardioplegic solution with PEP and ATP (group I) resulted in significantly reduced postischemic ventricular work and increased efflux of the creatine kinase isoenzyme MB (CK-MB). The same result was obtained when adenosine monophosphate (AMP) was added to the supplementation (group II). When guanosine monophosphate (GMP) was added instead of AMP (group III), the negative effects of PEP and ATP in the cardioplegic solution were partly abolished. Plain potassium cardioplegia, without additives, nevertheless gave the best results. There were no significant intergroup differences in the myocardial content of adenine nucleotides. The results contrasted with those in the previous study of more protracted ischemic trauma (30 min at 37 degrees C) and possible explanations are discussed.

Secretory granules and vesicles were demonstrated within human prostatic cells and in the acinar lumen. In size and ultrastructure the granules and vesicles were the same as those previously isolated from human prostatic fluid and seminal plasma. "Prostasomes" is suggested as the designation for these secretory granules and vesicles. As a rule they were found in storage vesicles within the secretory cells. Two mechanisms for translocation of the prostasomes from the cell interior to the acinar lumen are described. One mechanism involves exocytosis with binding of storage vesicles to, and fusion with the plasma membrane, resulting in release of prostasomes directly into the acinar lumen. The other mechanism implies displacement of storage vesicles in toto from the cell interior to the acinar lumen. This process differs from exocytosis and is here designated "diacytosis". Both phenomena appear to be of roughly equal frequency.

IN a previous paper1, the incorporation of radioactive inorganic phosphate into human red-cell ghosts was investigated in great detail. About 30 labelled phosphopeptides could be isolated, and their amino-acid patterns were determined. The rate of incorporation and the pH-dependence were also estimated. It was also shown2 that the 32P-incorporating membrane factor could be extracted and purified, and at least some part of the phospholipids also seemed to be metabolically active, since labelled phosphorylethanolamine and phosphorylcholine could be isolated. In addition, it was briefly mentioned that labelled trichloroacetic acid (TCA)-soluble adenosine triphosphate (ATP) seemed to be formed in the labelling process. In the TCA-insoluble material, labelled ultra-violet-absorbing substances were also present.

The homogeneity of an isolated membrane fraction of human erythrocytes was studied by density gradient centrifugation in silica solution. A main component (about 90% on a nitrogen basis) was obtained as a single band at a density of 1.08. When analyzed by electron microscopy, this component was found to consist of a membranous structure which, in contrast to isolated ghosts, appeared to be essentially devoid of cytoplasmic structures. The main component, obtained at a density of 1.08 of the gradient, was shown to contain glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and adenylte kinase. Thus, the present results strongly support the view that part of the glyceraldehyde-3-phosphate dehydrogenase, phophoglycerate kinase and adenylate kinase of the human erythrocyte is intimately associated with the membranous structure.

Zusammenfassung Es wird uber die Enzym-Aktivitat eines Mg2+ und Ca2+-abhangigen ATPase-Enzym-systems berichtet, die an eine subzellulare Organelle geknupft und in der Prostata-Flus-sigkeit und damit im Seminalplasma vorhanden ist. Die elektronmikroskopische Beobachtung des uber 12 h bei 40 000 × G zentrifugierten Sediments aus der Prostata-Flussigkeit als auch aus dem Seminalplasma zeigte sekretorische Granulae und Vesiculae, wobei die meisten Partikel von einer dreischichtigen Membran umschlossen waren. Andere jedoch zeigten eine funfschichtige Membran. Das ATPase-Enzymsystem wurde charakterisiert; die scheinbare Michaelis-Konstante (Km) sowie Vmax der Mg2+-abhangigen ATPase ergab 7,1 × 10−4 M bzw. 4,0 mikromol pro 0,1 ml Prostata-Flussigkeit und 5 Min. Die entsprechenden Werte der Ca2+-abhangigen ATPase ergaben 3,8 × 10−4 M und 3,2 mikromol pro 0,1 ml und 5 Min. Concanavalin A fordert bei niedriger Konzentration das Mg2+-abhangige ATPase-System, indem es bei entsprechender Bedingung die Ca2+-abhangige ATPase-Aktivitat hemmt. Mehrere divalente Kationen (Cu2+, Zn2+, Mn2+ und Sr2+) sowie La3+ waren unterschiedlich hemmend. Cytochalasin B wirkte sich bei 5 × 10−5 M zu etwa 30% auf die ATPase hemmend aus. Vinblastin und Colchicin zeigten geringen oder keinen Einflus auf das System. Lipolytische Stoffe wie Deoxycholat, Triton-X-100 und Dodecanylsulfat waren auf das ATPase-System alle gleich stark wirkungsvoll als hemmende Substanzen. Auch Reaktoren der Thiol-Gruppen wirkten im gewissen Ausmas hemmend. Bei Zusatz von Na+ und K+ zum ATPase-System wurde kein Aktivitatsanstieg erhalten. Weiterhin ergab Ouabain keine Hemmung. Die partikulare ATPase-enthaltende Flussigkeit stammt sicherlich aus der Prostata. Verschiedene denkbare Funktiorien dieses ATPase-Systems werden diskutiert.

Ninety homozygous patients with Hb S haemoglobinopathy from Sudan were reported with respect to clinical findings, haematological and serum parameters. For comparison, 27 Hb AS heterozygous subjects and 28 Hb AA controls were investigated also. The patients showed an extreme type of illness presenting with severe clinical signs such as dactylitis, liver enlargement and cardiac complications. There was marked haemolytic anaemia (mean haemoglobin 66 g/l). Some patients also presented with low serum (S)-iron levels, indicating iron deficiency. S-bilirubin, S-ASAT, S-ALAT, S-GT and S-urate were notably raised, most probably as a consequence of haemolysis with liver involvement. The patients had lower S-calcium levels when compared with the AS and AA subjects. This may suggest a possible role of HB S in the production of this feature.

Washed human erythrocytes were basically incubated in phosphate-buffered saline at 37 degrees C with different concentrations (1-5 mumol l-1) of the divalent cationophore A23187. This ionophore induced a decrease by about 75% of ATP content and energy charge (EC) and a concomitant increase in ADP and AMP contents in a dose-dependent fashion such that an inverse ATP/ADP relationship developed. EGTA at 1 mumol l-1 annihilated the effect of A23187 on energy status. When glucose was added to the basic incubation medium A23187 inclusion resulted in an elevated lactate production concomitantly with a partial restoration of EC. Introduction of Mg2+ to basic incubation medium containing glucose and the ionophore resulted in a sharp increase in lactate production with an energy state that was maintained at a control level. It is concluded that the low EC of erythrocytes obtained by this ionophore is the result of runaway ATPases dissociated from glycolysis. Since human erythrocytes are devoid of organelles other than the plasma membrane it is concluded that the ionophoric effect is exerted in the plasma membrane.

Prostasomes, which are prostate-derived organelles, were purified from human seminal plasma for inclusion in Earle's balanced salt solution (EBSS) medium with or without human serum albumin. These media were used for swim-up experiments and the subsequent analyses of sperm motility parameters at different incubation times. The yield of motile spermatozoa after swim-up in EBSS medium enriched with boiled prostasomes was increased by 32% compared with EBSS containing albumin. Native prostasomes were less active. Combinations of albumin and either prostasomes or boiled prostasomes significantly increased the recovery of motile spermatozoa and also increased the percentage of spermatozoa displaying progressive motility after 1 h of incubation. Media lacking albumin showed lower values regarding progressive motility after 22 h of incubation. A beneficial effect of prostasomes was noted on lateral head displacement and percentage of hyperactive spermatozoa during the first 6 h of incubation. These results suggest that inclusion of prostasomes, especially boiled prostasomes, in swim-up media may improve the recovery of hyperactive motile spermatozoa for up to 6 h in cases of established male factor infertility, and consequently increase the opportunities for fertilization.