Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Limiting factors for seed production in Cynoglossum officinale

Summary Over three years of study, small plants of Cynoglossum officinale consistently produced more flowers per unit of dry weight than large plants. In contrast to earlier results, weight of all seeds tended to increase more than proportional to size. As a result a positive correlation existed between seed set per flower and plant size. The correlation between the mean number of pollinator visits per flower and size was positive but not significant. In a field experiment we found that resources rather than pollen were limiting seed set. Thus, it is unlikely that enhanced pollination of the largest plants causes the size-dependency of seed set per flower. Alternative hypotheses are discussed briefly.Publication of the Meijendel Comité, New Series No. 96     

Picosecond Photochemistry of P700-Enriched and Vitamin K1-Depleted Photosystem I Particles Isolated from Spinach

Picosecond transient absorption changes, with a laser intensityas low as one photon absorbed per single reaction center, weremeasured with vitamin K₁-depleted and P700-enriched particleswhich were obtained by ether treatment of spinach PS-I particles.When P700 was in the oxidized state, a bleaching that correspondedto about one-seventh of the ground state absorption was observedjust after a laser flash (0 picosecond delay). A major partof the bleaching decayed with a lifetime of about 35 picoseconds,which corresponds to the relaxation of the excited antenna chl-ato the ground state. By contrast, when P700 was in the reducedstate, the bleaching observed at a 0 ps delay was broader, especiallyon the longer wavelength side than the ground state absorption,probably because of the generation of the excited state of P700.About one half of the bleaching decayed within 35 ps and theremaining half, which had a broad spectrum and a peak around682 nm, was conserved up to 2 ns. This long-lived bleachingprobes no picosecond decay of the radical pair P700⁺-A₀^–because electrons were not transferred from A₀¹ to A₁ in vitaminK₁-depleted particles. After addition of vitamin K₃, an analogof vitamin K₁, to the reduced particles, the bleaching around685 nm decayed successively with an apparent rate of about 150picosecond, while the bleaching around 700 nm was conservedfor up to 2 nanosecond. Thus, the bleaching remaining at 2 nsresembled the difference spectrum of P700, suggesting a subnanosecondquenching of A₀¹ by the externally added vitamin K₃. These observationssupport a recent proposal that the secondary electron acceptorA₁, in photosystem I, is vitamin K₁. ³Permanent address: Optics Laboratory, Korea Standards ResearchInstitute, Daedok Science Town, Chungnam 300-31, Korea. (Received October 24, 1988; Accepted April 14, 1989)     

Cytomegalovirus encephalitis: neuropathological comparison of the guinea pig model with the opportunistic infection in AIDS

The AIDS epidemic has transformed the importance of cytomegalovirus (CMV) as a pathogen for the adult human central nervous system (CNS). At autopsy, about 25 percent of AIDS cases have cytopathologic evidence of CNS infection by CMV. Since almost nothing is known of the host CNS-viral interactions, we have developed a laboratory model of CMV infection of the brain in the guinea pig. In the present paper, we review the syndromes of CMV infection of the human CNS and compare the neuropathological findings of the opportunistic CMV brain infection in AIDS with the model. Destructive meningoencephalitis, perivascular infiltrates, and subependymal inflammation are found in both, but the glial nodule is the most characteristic feature of each. Thus, we demonstrate that the model faithfully reflects the histopathology of the human disease. Furthermore, since we have found that CNS infection is achieved following systemic infection in the guinea pig, the model recapitulates the sequence of infection in humans.     

Effect of water activity on enzymatic synthesis of cephalexin

Summary In enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D--phenylglycine methyl ester (PGM) using an acylase fromXanthomonas citri, it was found that the synthetic activity and conversion yield were enhanced markedly by depressing the water activity (a _w ) of reaction system. This enhancement was probably resulted from the change of thermodynamic equilibrium and maximized at a range ofa _w from 0.96 to 0.97.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Two subunits of mannose permease, II-PMan and II-MMan, of Escherichia coli mediate coliphage N4 infection

Mutant strains of Escherichia coli that lack one or all of the intact components of mannose permease do not support the growth of phage N4. Complementation experiments using three recombinant plasmids containing DNA fragments coding for the subunits of mannose permease revealed that among the three component subunits, II-PMan and II-MMan alone are sufficient to confer N4 sensitivity.