Properties and characterization of a highly purified sarcoplasmic reticulum Ca2+-ATPase from dog cardiac and rabbit skeletal muscle

Sarcoplasmic reticulum (SR) Ca2+-ATPase was purified from dog cardiac and rabbit skeletal muscle using Triton X-100 at optimal ratios of 0.5 for cardiac and 0.5 to 1.0 for skeletal SR. The yields of Ca2+-ATPase were 4 to 5 and 1 to 2.2 mg/100 mg of cardiac and skeletal SR protein, respectively. The enzyme activities were 547 +/- 67 mumol ADP/mg/h for cardiac and 1192 +/- 172 mumol ADP/mg/h for skeletal Ca2+-ATPase. Removal of excess Triton X-100 increased the enzyme activities to 719 +/- 70 and 1473 +/- 206 mumol ADP/mg/h, respectively. The residual content of Triton X-100 for cardiac and skeletal Ca2+-ATPase was 20 and 5 mol/mol of enzyme, respectively. Maximum levels of phosphoenzyme were 4.4 +/- 0.2 and 5.6 +/- 0.6 nmol/mg in each case. A single protein band of 100 kDa was obtained for each purified Ca2+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparations were stable at -80 degrees C for 5 months in the presence of 1 mM Ca2+. The phospholipid content of the purified enzyme was 2-fold greater than that of native cardiac and skeletal SR microsomes. Repeated washing of the purified enzyme preparation did not alter the phospholipid content or the specific activities.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Anticonvulsant activity of glycylglycine and delta-aminovaleric acid: evidence for glutamine exchange in amino acid transport

We have proposed that glutamine serves in a facilitated diffusion process, mediated by the enzyme gamma-glutamyl transferase (gamma-glutamyl transpeptidase; gamma GT) and that it leaves the brain in exchange for entering amino acids. Glutamine is also a precursor of gamma-aminobutyric acid (GABA). Thus, providing an alternate substrate for gamma GT should spare brain glutamine, raise GABA, and cause an anticonvulsant effect. We have found that glycylglycine, the best-known substrate for gamma GT, and delta-aminovaleric acid (DAVA), a structural analog, have anticonvulsant activity in DBA/2J mice. Both compounds can decrease the incidence and severity of seizures induced by L-methionine-RS-sulfoximine or electroconvulsive shock. DAVA was also tested and found to be active against seizures caused by pentylenetetrazol or picrotoxin. [14C]DAVA entered the brain at the rate of 18.7 nmol/g/min. The activity of DAVA as a substrate of gamma GT was intermediate to that of glycylglycine and glutamine. Preliminary studies have shown that brain glutamine and perhaps GABA are elevated 3 h after administration of DAVA (7.5 mmol/kg). These findings support the theory that glutamine exchange plays a role in amino acid transport across the blood-brain barrier and suggests a new concept in anticonvulsant therapy.     

Intralipid and free plasmatic tryptophan in vitro

In an attempt to investigate the role of the lipidic emulsion Intralipid in the development of metabolic encephalopathy in a patient showing high free tryptophan levels, the relationship between lipidic emulsion and free tryptophan was examined in in vitro experiments. The addition of intralipid to normal serum produces an immediate increase in non-esterified fatty acids and a parallel rise in free tryptophan. Moreover, when serum with intralipid is incubated at 37 degrees C, the lipases release new non-esterified fatty acids and the free tryptophan increases proportionally. The non-esterified fatty acid content of intralipid was found to be 12 +/- 2 mEq X 1(-1). An inverse correlation was seen between free tryptophan and different serum albumin concentrations. It is concluded that intralipid causes an increase in free tryptophan levels. It is known that in vivo free tryptophan modulates 5-hydroxytryptamine synthesis and thus may be considered a possible causal agent for encephalopathy.     

Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm, Manduca sexta (L.)

The relationship between the ecdysteroid titre and eclosion hormone was explored for the pupal and adult ecdyses of Manduca sexta. Ecdysteroid treatment late during either moult caused a dosedependant delay in the time of ecdysis. Sensitivity to exogenous steroid treatment dropped off as the respective moults neared completion and in both cases coincided with the time of the low point in the endogenous ecdysteroid titre. It was concluded that an ecdysteroid decline is a normal prerequisite for the ecdyses of both stages. The steroid drop is important for two aspects of the eclosion hormone system: it causes target tissues to become sensitive to the peptide and it is a prerequisite for the subsequent release of eclosion hormone itself. Thus, the dual action of the declining ecdysteroid titre insures that when eclosion hormone is released, the tissues will be competent to respond to it.     

A unique DR-related B cell differentiation antigen

The Ia or class II molecules in both mouse and man are the basis for the genetic control of the immune response. In addition to DR, other class II antigens have been described in man. We describe a new human Ia antigen K19, recognized by three monoclonal antibodies (HK-9, HK-19, and HK-20). This antigen has the general biochemical characteristics of an Ia antigen but is different from a DR antigen. Further, this antigen is found only on mature B lymphocytes and not on monocytes and activated T cells. Thus, this antigen may represent a new Ia-like molecule that is preferentially expressed on mature B cells.     

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.     

Immunological evidence for two distinct chondroitin sulfate proteoglycan core proteins: Differential expression in cartilage matrix deficient mice

The expression and core protein structure of two proteoglycans, the major cartilage proteoglycan isolated from a rat chondrosarcoma and a small molecular weight chondroitin sulfate proteoglycan isolated from a rat yolk sac tumor, have been compared. The cartilage proteoglycan was not detectable in the cartilage tissue of cartilage matrix deficient ($\text{cmd}\text{cmd}$) neonatal mice by immunofluorescence, but the cmd cartilage did react with antibodies against the core protein of the yolk sac tumor proteoglycan. Radioimmunoassays showed that the core proteins of these proteoglycans are not cross-reactive with each other. Analysis of the core proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after chondroitinase ABC treatment of the proteoglycan revealed a large difference in their sizes. The cartilage proteoglycan core protein had a molecular weight of about 200,000 while the yolk sac tumor proteoglycan core protein migrated with an apparent molecular weight of about 20,000. In addition, the cultured yolk sac tumor cells that make the small proteoglycan did not react with antiserum against the cartilage proteoglycan. These results indicate that the proteoglycan isolated from the yolk sac tumor is similar to the small chondroitin sulfate proteoglycan species found in cartilage and support the existence of at least two dissimilar and genetically independent chondroitin sulfate proteoglycan core proteins.     

Direct effects on the membrane potential due to "pumps" that transfer no net charge

The effects of active ionic transport are included in the derivation of a general expression for the zero current membrane potential. It is demonstrated that an active transport system that transfers no net charge (nonrheogenic) may, nevertheless, directly alter the membrane potential. This effect depends upon the exchange of matter within the membrane between the active and passive diffusion regimes. Furthermore, in the presence of such exchange, the transmembrane active fluxes measured by the usual techniques and the local pumped fluxes are not identical. Several common uses of the term “electrogenic pump” are thus shown to be inconsistent with each other. These inconsistencies persist when the derivation is extended to produce a Goldman equation modified to account for active transport; however, that equation is shown to be limited by less narrow constraints on membrane heterogeneity and internal electric field than those previously required. In particular, it is applicable to idealized mosaic membranes limited by these requirements.