Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.     

The nutritional ecology of the ctenophore Bolinopsis vitrea: comparisons with Mnemiopsis mccradyi from the same region

Bolinopsis vitrea is a warm water lobate ctenophore which doesnot overlap in its distribution with Mnemiopsis mccradyi incontiguous waters. We examined its feeding ecology on a seriesof cruises. B. virrea ingested increasingly more prey at higherfood concentrations (2–100 prey l^–1) but feedingeffort (clearance rate) decreased with increasing food availability.On a dry weight basis, smaller tentaculate Bolinopsis ingestedseveral times more than larger lobates, but based on carbonweight, specific ingestion was fairly uniform over the entiresize range investigated (6–60 mm total length). Bolinopsiscollected during the daytime in the Bahamas rarely had morethan three prey items in their guts. These results and laboratorymeasurements of digestion times (av. = 1.9 h) allowed computationof daily rations, which could not account for the metabolicrequirement as measured on the same cruises. Results of feedingexperiments, however, implied that prey densities in excessof 11^–1 were sufficient to sustain a growing populationof Bolinopsis. Prey concentrations about an order of magnitudehigher were required for M. mccradyi based on similar experiments.These results were in general agreement with observed densitiesand distributions of ctenophores and their zooplankton preyin the Bahamas and coastal South Florida.     

Proteins of the human placental microvillar cytoskeleton. alpha-Actinin.

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.     

A detailed examination of the iodination of beta-galactosidase: stoichiometric inactivation by nonspecific iodination

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.     

Control of the activation/inactivation of pyruvate, Pi dikinase from the C4 plant maize by adenylate energy charge, pyruvate, and analogs of pyruvate

Pyruvate, Pi dikinase, which is localized in the mesophyll chloroplasts of C4 plants, requires a high adenylate energy charge for conversion of the enzyme from the inactive to the active form. The inactivation process is favored by a low energy charge, being maximal at values below 0.7. Pyruvate and analogs of pyruvate, oxamate and oxalate, strongly inhibit the inactivation process at millimolar levels. The results suggest that light activation of the enzyme in vivo may be mediated by an increased adenylate energy charge in the chloroplast. Pyruvate may allow a higher steady-state level of activation to be achieved in vivo by inhibiting inactivation.     

Cultural Aspects of Health and Illness Behavior in Hospitals

Health care attitudes reflect the basic world view and values of a culture, such as how we relate to nature, other people, time, being, society versus community, children versus elders and independence versus dependence. Illness behavior determines who is vulnerable to illness and who agrees to become a patient—since only about one third of the ill will see a physician. Cultural values determine how one will behave as a patient and what it means to be ill and especially to be a hospital patient. They affect decisions about a patient''s treatment and who makes the decisions. Cultural differences create problems in communication, rapport, physical examination and treatment compliance and follow through. The special meaning of medicines and diet requires particular attention. The perception of physical pain and psychologic distress varies from culture to culture and affects the attitudes and effectiveness of care-givers as much as of patients. Religious beliefs and attitudes about death, which have many cultural variations, are especially relevant to hospital-based treatment. Linguistic and cultural interpreters can be essential; they are more available than realized, though there are pitfalls in their use. Finally, one must recognize that individual characteristics may outweigh the ethnic and that a good caring relationship can compensate for many cultural missteps.     

Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.